KR102565305B1 - Composition for preparing 3-hydroxy phlorizin - Google Patents

Composition for preparing 3-hydroxy phlorizin Download PDF

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KR102565305B1
KR102565305B1 KR1020210085030A KR20210085030A KR102565305B1 KR 102565305 B1 KR102565305 B1 KR 102565305B1 KR 1020210085030 A KR1020210085030 A KR 1020210085030A KR 20210085030 A KR20210085030 A KR 20210085030A KR 102565305 B1 KR102565305 B1 KR 102565305B1
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윤철호
응우옌응억안
카오응억딴
강형식
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전남대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)

Abstract

본 발명의 일 실시예는 CYP102A1의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 3-히드록시 플로리진 제조용 조성물, 키트, 및 이를 이용하는 3-히드록시 플로리진의 제조방법을 제공한다.One embodiment of the present invention provides a composition and kit for preparing 3-hydroxy phlorizin comprising at least one enzyme selected from the group consisting of mutants of CYP102A1, and a method for preparing 3-hydroxy phlorizin using the same.

Description

3-히드록시 플로리진 제조용 조성물{COMPOSITION FOR PREPARING 3-HYDROXY PHLORIZIN}Composition for preparing 3-hydroxy phlorizin {COMPOSITION FOR PREPARING 3-HYDROXY PHLORIZIN}

본 발명은 박테리아 시토크롬 P450을 이용하여 플로리진으로부터 그의 대사체인 3-히드록시 플로리진을 제조하기 위한 신규한 조성물에 관한 것이다.The present invention relates to a novel composition for the production of its metabolite, 3-hydroxyphlorizin, from phlorizin using bacterial cytochrome P450.

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플로리진(phlorizin)은 사과나무에 풍부하게 포함된 플로레틴(phloretin)의 자연적으로 발생하는 글루코사이드인 전형적인 디하이드로칼콘 화합물이다. 이러한 플로리진은 여러 분야에서 응용 가능한 강력한 항산화 활성 등의 특성을 가지고 있어, 의료, 식품 등의 분야에서 널리 활용될 수 있는 가능성을 가지고 있다.Phlorizin is a typical dihydrochalcone compound, a naturally occurring glucoside of phloretin, which is abundant in apple trees. Such phlorizin has properties such as strong antioxidant activity that can be applied in various fields, and has the potential to be widely used in fields such as medicine and food.

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최근 연구에 따르면 플로리진을 장기간 사용하면 당뇨병을 갖는 동물 모델의 혈당 수치와 지질 대사가 감소될 수 있음이 보고되었다. 상기 플로리진은 또한 소장 점막(SGLT1)과 신장 근위 세뇨관(SGLT2)에 위치한 나트륨-포도당 결합체(SGLT)에 대한 특이적이고 경쟁적인 억제제로 밝혀졌다. 따라서 신장에서의 포도당 재흡수 및 장에서의 포도당 흡수를 차단하여 고혈당증을 개선할 수 있다. 또한 플로리진은 장내 미생물총의 구조를 변화시킴으로써 인슐린 감수성을 향상시킬 수 있다. 또한 항산화 활성을 가지고 있다. Recent studies have reported that long-term use of phlorizin can reduce blood glucose levels and lipid metabolism in animal models with diabetes. The phlorizin was also found to be a specific and competitive inhibitor of sodium-glucose conjugates (SGLT) located in the small intestine mucosa (SGLT1) and the proximal tubule of the kidney (SGLT2). Therefore, hyperglycemia can be improved by blocking glucose reabsorption in the kidney and glucose absorption in the intestine. Phlorizin can also improve insulin sensitivity by altering the structure of the gut microbiota. It also has antioxidant activity.

한편, 3-히드록시 플로리진(3-hydroxyphlorizin)은 주로 malus 식물에서 발견되는 천연 배당체로, 용매 추출 방법을 통해 정제할 수 있으나, 이의 화학 합성 방법은 아직 보고되지 않고 있으며, 이의 생리적, 약리적 활성은 아직 광범위하게 연구되고 있지 않은 실정이다. 전통적인 화학적 수단으로 플로리진의 유도체를 선택적이고 효율적으로 합성하기가 어렵기 때문에 이에 대한 대안이 필요한 실정이다.On the other hand, 3-hydroxyphlorizin is a natural glycoside found mainly in malus plants and can be purified through solvent extraction, but its chemical synthesis method has not yet been reported, and its physiological and pharmacological activities has not yet been extensively studied. Since it is difficult to selectively and efficiently synthesize derivatives of phlorizin by traditional chemical means, an alternative to this is required.

Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH, Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol. Bioeng. 111:1313-1322, 2014 Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH, Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol. Bioeng. 111:1313-1322, 2014 Le TK, Jang HH, Nguyen HT, Doan TT, Lee GY, Park KD, Ahn T, Joung YH, Kang HS, Yun CH, Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3. Enzyme Microb Technol. 97:34-42, 2017 Le TK, Jang HH, Nguyen HT, Doan TT, Lee GY, Park KD, Ahn T, Joung YH, Kang HS, Yun CH, Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3. Enzyme Microb Technol. 97:34-42, 2017

본 발명의 일 과제는 플로리진의 대사산물인 3-히드록시 플로리진을 대량으로 생산할 수 있는 박테리아 시토크롬 P450 효소 및 이의 변이체를 포함하는 신규 조성물을 제공하는 것이다.One object of the present invention is to provide a novel composition comprising a bacterial cytochrome P450 enzyme capable of mass-producing 3-hydroxy phlorizin, a metabolite of phlorizin, and variants thereof.

본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 기술적 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The technical problem to be achieved by the present invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

상기 기술적 과제를 달성하기 위하여, 본 발명의 일 실시예는 CYP102A1의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 3-히드록시 플로리진(3-hydroxyphlorizin) 제조용 조성물로서, 상기 CYP102A1는 서열번호 1의 아미노산 서열을 포함하고, 상기 CYP102A1의 돌연변이체는 상기 서열번호 1로부터 치환되는 아미노산이 In order to achieve the above technical problem, one embodiment of the present invention is a composition for preparing 3-hydroxyphlorizin comprising at least one enzyme selected from the group consisting of mutants of CYP102A1, wherein the CYP102A1 is a sequence It includes the amino acid sequence of SEQ ID NO: 1, and the mutant of CYP102A1 has an amino acid substituted from SEQ ID NO: 1

R47L/F81I/F87V/E143G/L188Q/N213S/E267V, R47L/F81I/F87V/E143G/L188Q/N213S/E267V;

F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R;

D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V, D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V;

F42L/R47L/F81I/F87V/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V;

T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V;

R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R 및 R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R and

F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R으로 구성되는 군으로부터 선택되는 하나 이상을 포함하는 3-히드록시 플로리진 제조용 조성물을 제공한다.F11Y / R47L / D68G / F81I / F87V / E143G / L188Q / E267V / H408R provides a composition for preparing 3-hydroxy phlorizin containing at least one selected from the group consisting of.

본 발명의 실시예에 있어서, 상기 CYP102A1의 돌연변이체는 상기 서열번호 1로부터 치환되는 아미노산이 A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E 인 것을 추가로 포함하는 것을 특징으로 하는 3-히드록시 플로리진 제조용 조성물일 수 있다.In an embodiment of the present invention, in the mutant of CYP102A1, the amino acids substituted from SEQ ID NO: 1 are A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S / S954N / M967V / Q981R / A1008D / H1021Y / Q1022E may be a composition for preparing 3-hydroxy phlorizin, characterized in that it further comprises.

상기 기술적 과제를 달성하기 위하여, 본 발명의 다른 실시예는 상기 3-히드록시 플로리진 제조용 조성물을 포함하는 3-히드록시 플로리진의 생산용 키트를 제공한다.In order to achieve the above technical problem, another embodiment of the present invention provides a kit for producing 3-hydroxy phlorizin comprising the composition for preparing 3-hydroxy phlorizin.

상기 기술적 과제를 달성하기 위하여, 본 발명의 또 다른 실시예는 상기 3-히드록시 플로리진 제조용 조성물과 플로리진을 반응시키는 단계를 포함하는 3-히드록시 플로리진의 제조방법을 제공한다.In order to achieve the above technical problem, another embodiment of the present invention provides a method for producing 3-hydroxy phlorizin comprising the step of reacting the composition for preparing 3-hydroxy phlorizin with phlorizin.

본 발명의 실시예에 있어서, NADPH-생성 시스템을 추가하는 단계를 더 포함하는 것을 특징으로 3-히드록시 플로리진의 제조방법일 수 있다.In an embodiment of the present invention, it may be a method for producing 3-hydroxy phlorizin, characterized by further comprising the step of adding a NADPH-producing system.

본 발명의 일 실시예의 박테리아 시토크롬 P450, 즉, CYP102A1의 돌연변이체를 이용하여 플로리진의 대사산물인 3-히드록시 플로리진을 특이적으로 대량 생산할 수 있다. 상기 3-히드록시 플로리진은 화학적 합성방법이 보고된 바 없어, 본 발명의 일 실시예는 이의 대량 생산을 위한 대안을 제공할 수 있다.3-hydroxyphlorizin, a metabolite of phlorizin, can be specifically mass-produced using the mutant of bacterial cytochrome P450, ie, CYP102A1, according to one embodiment of the present invention. Since no chemical synthesis method has been reported for the 3-hydroxyphlorizin, an embodiment of the present invention can provide an alternative for its mass production.

본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 특허청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

도 1은 본 발명의 일 실시예에 따른 CYP102A1 야생형 및 이의 돌연변이체에 의한 플로리진의 대사산물 생성속도를 나타낸다.
도 2는 CYP102A1 돌연변이에 의해 형성된 플로리진과 그 생성물의 HPLC 크로마토그램을 나타낸다.
도 3은 CYP102A1 M371 돌연변이에 의해 생성된 플로리진 및 그 생성물의 LC-MS 분석을 나타낸다.
도 4는 CYP102A1 M371에 의한 플로리진과 주요 산물의 HPLC 크로마토그램을 나타낸다.
도 5는 CYP102A1 M371 및 β-글루코시다아제에 의해 생산된 플로리진과 그 제품의 LC-MS/MS 분석을 나타낸다.
도 6은 NADPH 존재 하에 M10과 함께 배양된 플로레틴의 MS/MS 분석을 나타낸다.
도 7은 CYP102A1 돌연변이에 의한 플로리진 히드록실화의 TTN을 나타낸다.
도 8은 플로리진을 사용한 CYP102A1의 스펙트럼 결합 적정을 나타낸다.
도 9는 야생형 CYP102A1의 아미노산 서열을 나타낸다.
도 10은 야생형 CYP102A1의 돌연변이체 M16의 아미노산 서열을 나타낸다.
도 11은 야생형 CYP102A1의 돌연변이체 M16V2의 아미노산 서열을 나타낸다.
도 12는 야생형 CYP102A1의 돌연변이체 M179의 아미노산 서열을 나타낸다.
도 13은 야생형 CYP102A1의 돌연변이체 M221의 아미노산 서열을 나타낸다.
도 14는 야생형 CYP102A1의 돌연변이체 M371의 아미노산 서열을 나타낸다.
도 15는 야생형 CYP102A1의 돌연변이체 M524의 아미노산 서열을 나타낸다.
도 16은 야생형 CYP102A1의 돌연변이체 M634의 아미노산 서열을 나타낸다.
도 17은 야생형 CYP102A1의 돌연변이체 M788의 아미노산 서열을 나타낸다.
도 18은 야생형 CYP102A1의 돌연변이체 M850의 아미노산 서열을 나타낸다.1 shows the rate of metabolite production of phlorizin by CYP102A1 wild type and mutants thereof according to an embodiment of the present invention.
Figure 2 shows HPLC chromatograms of phlorizin and its products formed by CYP102A1 mutation.
Figure 3 shows LC-MS analysis of phlorizin and its products produced by the CYP102A1 M371 mutation.
Figure 4 shows the HPLC chromatogram of phlorizin and the main product by CYP102A1 M371.
Figure 5 shows LC-MS/MS analysis of phlorizin and its products produced by CYP102A1 M371 and β-glucosidase.
6 shows MS/MS analysis of phloretin incubated with M10 in the presence of NADPH.
Figure 7 shows the TTN of phlorizin hydroxylation by CYP102A1 mutation.
Figure 8 shows a spectral binding titration of CYP102A1 with phlorizin.
9 shows the amino acid sequence of wild-type CYP102A1.
10 shows the amino acid sequence of mutant M16 of wild type CYP102A1.
11 shows the amino acid sequence of mutant M16V2 of wild type CYP102A1.
Figure 12 shows the amino acid sequence of mutant M179 of wild type CYP102A1.
13 shows the amino acid sequence of mutant M221 of wild type CYP102A1.
14 shows the amino acid sequence of mutant M371 of wild type CYP102A1.
15 shows the amino acid sequence of mutant M524 of wild type CYP102A1.
16 shows the amino acid sequence of mutant M634 of wild type CYP102A1.
17 shows the amino acid sequence of mutant M788 of wild type CYP102A1.
18 shows the amino acid sequence of mutant M850 of wild type CYP102A1.

이하에서는 첨부한 도면을 참조하여 본 발명을 설명하기로 한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며, 따라서 여기에서 설명하는 실시예로 한정되는 것은 아니다. 그리고 도면에서 본 발명을 명확하게 설명하기 위해서 설명과 관계없는 부분은 생략하였으며, 명세서 전체를 통하여 유사한 부분에 대해서는 유사한 도면 부호를 붙였다.Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in many different forms and, therefore, is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

명세서 전체에서, 어떤 부분이 다른 부분과 "연결(접속, 접촉, 결합)"되어 있다고 할 때, 이는 "직접적으로 연결"되어 있는 경우뿐 아니라, 그 중간에 다른 부재를 사이에 두고 "간접적으로 연결"되어 있는 경우도 포함한다. 또한 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 구비할 수 있다는 것을 의미한다.Throughout the specification, when a part is said to be "connected (connected, contacted, combined)" with another part, this is not only "directly connected", but also "indirectly connected" with another member in between. "Including cases where In addition, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

본 명세서에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Terms used in this specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

이하 첨부된 도면을 참고하여 본 발명의 실시예를 상세히 설명하기로 한다.Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

본 발명의 일 양태는 박테리아 시토크롬 P450의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 3-히드록시 플로리진 제조용 조성물을 제공한다.One aspect of the present invention provides a composition for preparing 3-hydroxy phlorizin comprising at least one enzyme selected from the group consisting of mutants of bacterial cytochrome P450.

상기 3-히드록시 플로리진 제조용 조성물에 포함된 박테리아 시토크롬 P450 효소 및 이의 돌연변이체는, 하기 반응식 1과 같이, 플로리진을 기질로 하여 대사물질인 3-히드록시 플로리진을 선택적이며 안정적이고 고효율로 대량 생산할 수 있다.The bacterial cytochrome P450 enzyme and its mutants included in the composition for preparing 3-hydroxyphlorizin selectively, stably and efficiently converts the metabolite 3-hydroxyphlorizin using phlorizin as a substrate, as shown in Scheme 1 below. can be mass produced.

[반응식 1][Scheme 1]

상기 박테리아 시토크롬 P450 효소는 박테리아 시토크롬 P450 BM3인 CYP102A1일 수 있다. 상기 CYP102A1은 서열번호 1의 아미노산 서열을 가질 수 있다.The bacterial cytochrome P450 enzyme may be bacterial cytochrome P450 BM3, CYP102A1. The CYP102A1 may have the amino acid sequence of SEQ ID NO: 1.

상기 CYP102A1의 돌연변이체는 야생형 CYP102A1을 기준으로 아미노산 서열의 자연적 또는 인위적인 치환, 결실, 부가 및/또는 삽입에 의하여 변화된 서열을 가지는 서열의 일부 또는 전체를 의미할 수 있다. 상기 치환되는 아미노산은 아래 분류된 바와 같이 치환될 아미노산과 유사한 성질을 갖는 것으로 치환될 수 있다. 예를 들어, 알라닌, 발린, 류신, 이소류신, 프롤린, 메티오닌, 페닐알라닌 및 트립토판은 모두 비극성 아미노산으로 분류되며, 이들은 서로 비슷한 성질을 갖는다. 전하를 띄지 않는 아미노산으로는 글리신, 세린, 트레오닌, 시스테인, 티로신, 아스파라진, 글루타민을 포함하며, 산성 아미노산으로는 아스파르산, 글루타민산을 염기성 아미노산으로는 리신, 아르기닌, 히스티딘을 포함한다. 예를 들어, 야생형 CYP102A의 아미노산 서열을 기준으로 점 돌연변이가 일어난 개체나 CYP102A1의 서로 다른 도메인의 융합 키메라를 포함할 수 있다.The mutant of CYP102A1 may refer to part or all of a sequence having a sequence changed by natural or artificial substitution, deletion, addition, and/or insertion of an amino acid sequence based on wild-type CYP102A1. The amino acid to be substituted may be substituted with one having properties similar to the amino acid to be substituted, as classified below. For example, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan are all classified as non-polar amino acids, and they have similar properties to each other. Uncharged amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, acidic amino acids include aspartic acid and glutamic acid, and basic amino acids include lysine, arginine, and histidine. For example, it may include individuals in which point mutations have occurred based on the amino acid sequence of wild-type CYP102A or fusion chimeras of different domains of CYP102A1.

상기 CYP102A1의 돌연변이체는 공지의 돌연변이 유도 방법으로 제조할 수 있다. 상기 돌연변이 유도 방법은, 예를 들어, 결실-돌연변이 방법, PCT 방법, 쿤켈법, 점 돌연변이 방법, DNA 셔플링 방법, StEP(staggered extension process), 오류유발(error-prone) PCR 방법 등을 포함할 수 있으나, 이에 한정되는 것은 아니다.The mutant of CYP102A1 can be prepared by a known mutagenesis method. The mutagenesis method may include, for example, a deletion-mutation method, a PCT method, a Kunkel method, a point mutation method, a DNA shuffling method, a staggered extension process (StEP), an error-prone PCR method, and the like. It may, but is not limited thereto.

상기 CYP102A1의 돌연변이체는 야생형 CYP102A1의 아미노산 서열과 50% 이상, 예를 들어, 70% 이상, 예를 들어, 90% 이상의 상동성을 가지는 아미노산 서열을 포함할 수 있다.The mutant of CYP102A1 may include an amino acid sequence having 50% or more, eg, 70% or more, eg, 90% or more homology to the amino acid sequence of wild-type CYP102A1.

본 발명에서, 키메라(chimera)는 서로 다른 두 개 이상의 결합 도메인(domain)을 포함할 수 있다. 두 결합 도메인은 상이한 야생형 단백질 또는 돌연변이체에서 유래할 수 있고, 두 결합 도메인은 동일한 야생형 단백질 또는 돌연변이체에서 유래할 수도 있다. 키메라 CYP102A1은 야생형 CYP102A1 돌연변이체의 헴 영역(heme domain)과 야생형 CYP102A1의 자연적 변형체(nature variants)의 환원효소 영역(reductase domain)을 융합한 CYP102A1 키메라 및 CYP102A1 키메라의 점 돌연변이체를 포함할 수 있다.In the present invention, a chimera may include two or more different binding domains. The two binding domains may be from different wild-type proteins or mutants, and the two binding domains may be from the same wild-type protein or mutants. The chimera CYP102A1 may include a CYP102A1 chimera in which a heme domain of a wild-type CYP102A1 mutant and a reductase domain of a natural variant of wild-type CYP102A1 are fused, and point mutants of the CYP102A1 chimera.

상기 자연적 변형체는 자연계에서 채집되어 기탁된 바실러스 종(Bacillus sp.)의 CYP102A1 유전자의 염기서열을 확인한 결과 기존에 알려진 CYP102A1외에 자연계에 존재하는 변이체를 의미한다. 키메라 CYP102A1은 야생형 CYP102A1의 점 돌연변이체들을 대장균에서 대량 발현시켜 돌연변이체들 중 기질에 대한 촉매 활성이 큰 것을 선택하고, 선택한 CYP102A1 돌연변이체의 헴 영역과 야생형 CYP102A1의 자연적 변형체의 환원효소 영역을 융합하여 제조할 수 있다. 그리고 제조한 키메라 CYP102A1의 점 돌연변이체들을 대장균에서 대량 발현시켜 촉매 활성이 큰 것을 선택하여 키메라 CYP102A1의 돌연변이체를 선정할 수 있다.The natural variant means a variant that exists in nature other than the previously known CYP102A1 as a result of confirming the nucleotide sequence of the CYP102A1 gene of Bacillus sp., which was collected and deposited in nature. Chimera CYP102A1 is produced by mass-expressing wild-type CYP102A1 point mutants in Escherichia coli, selecting those with high catalytic activity for the substrate among the mutants, and fusing the heme region of the selected CYP102A1 mutant with the reductase region of the natural variant of wild-type CYP102A1. can be manufactured In addition, the chimeric CYP102A1 mutants can be selected by mass-expressing the prepared chimeric CYP102A1 point mutants in Escherichia coli and selecting those having high catalytic activity.

상기 CYP102A1의 돌연변이체는 '(변이가 일어나기 전 아미노산), (아미노산의 위치), (변이가 일어나 치환된 아미노산)'의 순서로 표시될 수 있다. 예를 들어, R47L은 야생형 CYP102A1의 아미노산 서열인 서열번호 1의 47번째 아미노산인 아르기닌(R)이 류신(L)으로 자연적 또는 인위적으로 변이 유도에 의해 치환된 CYP102A1 돌연변이체를 의미한다. 돌연변이체에서 변이에 의해 치환된 아미노산이 하나 이상인 경우 '/'로 나타낼 수 있다. 예를 들어, R47L/F81I/F87V는 서열번호 1의 47번째 아미노산인 아르기닌(R)이 류신(L)으로 치환되고, 서열번호 1의 81번째 아미노산인 페닐알라닌(F)이 이소류신(I)으로 치환되고, 서열번호 1의 87번째 아미노산인 페닐알라닌(F)이 발린(V)으로 치환된 야생형 CYP102A1의 돌연변이체를 의미한다. 이와 같은 표시 방법은 본 발명에서 키메라 CYP102A1 또는 키메라 CYP102A1의 돌연변이체 변이를 나타내는데 사용할 수 있다.The mutants of CYP102A1 may be displayed in the order of '(amino acid before mutation), (position of amino acid), (amino acid substituted after mutation)'. For example, R47L refers to a CYP102A1 mutant in which arginine (R), the 47th amino acid of SEQ ID NO: 1, which is the amino acid sequence of wild-type CYP102A1, is naturally or artificially substituted with leucine (L) by mutagenesis. When one or more amino acids are substituted by mutation in the mutant, it can be represented by '/'. For example, in R47L/F81I/F87V, the 47th amino acid of SEQ ID NO: 1, arginine (R) is substituted with leucine (L), and the 81st amino acid of SEQ ID NO: 1, phenylalanine (F) is substituted with isoleucine (I). and a mutant of wild-type CYP102A1 in which phenylalanine (F), the 87th amino acid of SEQ ID NO: 1, is substituted with valine (V). Such a display method can be used to indicate chimeric CYP102A1 or mutant mutants of chimeric CYP102A1 in the present invention.

상기 야생형 CYP102A1의 돌연변이체는 야생형 CYP102A1의 점 돌연변이체일 수 있다. 예를 들어, 야생형 CYP102A1의 점 돌연변이체인 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/N213S/E267V, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R, D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V, R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R 및/또는 F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R의 야생형 CYP102A1의 돌연변이체일 수 있다.The wild-type CYP102A1 mutant may be a point mutant of wild-type CYP102A1. For example, R47L/F81I/F87V/E143G/L188Q/N213S/E267V, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R, D23G/R47L/ F81I/F87V /F107L/D136G/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V, R47L/F81I/ F87V/K113N/E143G /L188Q/E267V/N319D/L347I/S383R and/or F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R may be mutants of wild type CYP102A1.

상기 야생형 CYP102A1의 돌연변이체는 야생형 CYP102A1의 점 돌연변이체의 햄 영역과 야생형 CYP102A1의 자연적 변형체의 환원 영역을 융합한 키메라 CYP102A1를 포함할 수 있다. 예를 들어, 야생형 CYP102A1의 점 돌연변이체인 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/N213S/E267V, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R, D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V, R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R 및/또는 F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R의 헴 영역과 야생형 CYP102A1의 자연적 변형체의 환원 영역인 A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E이 융합된, 야생형 CYP102A1의 돌연변이체일 수 있다.The wild-type CYP102A1 mutant may include a chimera CYP102A1 obtained by fusing a ham region of a point mutant of wild-type CYP102A1 with a reduced region of a natural variant of wild-type CYP102A1. For example, R47L/F81I/F87V/E143G/L188Q/N213S/E267V, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R, D23G/R47L/ F81I/F87V /F107L/D136G/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V, R47L/F81I/ F87V/K113N/E143G The heme region of /L188Q/E267V/N319D/L347I/S383R and/or F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R and A474V/E558D/T664A, the reducing region of a natural variant of wild-type CYP102A1 /P675L /A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E may be a fused mutant of wild-type CYP102A1.

이상의 야생형 CYP102A1의 돌연변이체에 대한 설명을 종합하면, 야생형 CYP102A1의 돌연변이체는 야생형 CYP102A1의 점 돌연변이체, 야생형 CYP102A1의 자연적 변이체, 야생형 CYP102A1의 점 돌연변이체의 헴 영역과 야생형 CYP102A1의 자연적 변이체의 환원효소 영역을 융합한 키메라 CYP102A1 및 키메라 CYP102A1의 점 돌연변이체를 포함할 수 있다.Summarizing the description of the wild-type CYP102A1 mutants above, the wild-type CYP102A1 mutants are a point mutant of wild-type CYP102A1, a natural variant of wild-type CYP102A1, a heme region of a point mutant of wild-type CYP102A1, and a reductase of a natural variant of wild-type CYP102A1. chimeric CYP102A1 fused regions and point mutants of chimeric CYP102A1.

예를 들어, 야생형 CYP102A1의 돌연변이체는 서열번호 1로 표시되는 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/N213S/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F42L/R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E 및/또는 F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E로 구성된 군으로부터 선택되는 하나 이상일 수 있다.For example, mutants of wild-type CYP102A1 are R47L/F81I/F87V/E143G/L188Q/N213S/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E of wild-type CYP102A1 represented by SEQ ID NO: 1 /R825S /R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E5 58D/T664A/P675L/A678E/E687A /A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, D23G/R47L/F81I/F87V/F107L/D136G/E1 43G/L188Q/E267V/A474V/E558D /T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F42L/R47L/ F81I/F87V/E143G/L188Q/E267V /A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022 E, T1S/R47L/F81I/F87V/E143G /T146A/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A 1008D/H1021Y/Q1022E, R47L/F81I /F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E8 87G/P894S/S954N/M967V/Q981R /A1008D/H1021Y/Q1022E and/or F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R 825S/R836H/E870N/I881V / E887G / P894S / S954N / M967V / Q981R / A1008D / H1021Y / Q1022E may be one or more selected from the group consisting of.

야생형 CYP102A1, 야생형 CYP102A1의 점 돌연변이체, 키메라 CYP102A1 및 키메라 CYP102A1의 점 돌연변이체는 본 발명이 속하는 분야에서 공지된 방법으로 제조할 수 있다. 예를 들어, 유전공학적 기법, 고체상 기술을 사용하는 펩티드 합성방법(Merrifield, J. Am. Chem. Soc., 85 : 2149-2154(1963) 참조) 또는 본 발명의 일 실시예에 따른 효소를 적당한 펩티다제로 절단하는 등의 방법으로 제조할 수 있다.Wild-type CYP102A1, point mutants of wild-type CYP102A1, chimeric CYP102A1, and point mutants of chimeric CYP102A1 can be prepared by methods known in the art. For example, a peptide synthesis method using a genetic engineering technique, a solid-phase technique (see Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)), or an enzyme according to an embodiment of the present invention is suitable. It can be produced by a method such as digestion with peptidase.

상기 효소는 천연 단백질로서 제조할 수 있고, 야생형 CYP102A1, 야생형 CYP102A1의 점 돌연변이체, 키메라 CYP102A1 및 키메라 CYP102A1의 점 돌연변이체를 암호화하는 DNA로 형질전환시킨 세포를 배양하여 회수하는 재조합 방법에 의해 제조할 수도 있다. 구체적으로, 본 발명의 효소를 암호화하는 핵산 분자를 적절한 발현 벡터로 삽입하고 발현 벡터를 적합한 세포로 전달하여 만든 형질 전환체를 배양한 후 형질전환체에 의해 발현된 효소를 정제함으로써 본 발명의 효소를 생산할 수 있다.The enzyme can be produced as a natural protein, and can be produced by a recombinant method in which cells transformed with DNA encoding wild-type CYP102A1, point mutants of wild-type CYP102A1, chimeric CYP102A1, and point mutants of chimeric CYP102A1 are cultured and recovered. may be Specifically, the enzyme of the present invention is obtained by inserting a nucleic acid molecule encoding the enzyme of the present invention into an appropriate expression vector, culturing a transformant made by transferring the expression vector to a suitable cell, and then purifying the enzyme expressed by the transformant. can produce

상기 벡터는 플라스미드(plasmid), 코스미드(cosmid), 바이러스 입자 또는 파지(phage) 형태일 수 있다. 벡터 내의 DNA를 클로닝하거나 발현시키기 위한 숙주세포는 원핵세포, 효모, 고등진핵세포를 포함할 수 있다. 배지, 온도, pH 등의 배양 조건은 본 발명이 속하는 분야에서 과도한 실험 없이 적합하게 선택할 수 있다. 예를 들어, 세포배양의 생산성을 최대로 하기 위한 원리, 프로토콜, 테크닉 등은 여러 공지된 방법을 참조할 수 있다(Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) 등).The vector may be in the form of a plasmid, cosmid, viral particle or phage. Host cells for cloning or expressing the DNA in the vector may include prokaryotic cells, yeast, and higher eukaryotic cells. Culture conditions such as medium, temperature, pH, etc. can be appropriately selected without undue experimentation in the field to which the present invention belongs. For example, principles, protocols, techniques, etc. for maximizing the productivity of cell culture may refer to several known methods (Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)). etc).

상기 발현 및 클로닝 벡터는 일반적으로 mRNA 합성을 유도하는 야생형 CYP102A1, 야생형 CYP102A1의 점 돌연변이체, 키메라 CYP102A1 및 키메라 CYP102A1의 점 돌연변이체를 코딩하는 핵산 서열에 작동 가능하도록 연결된 프로모터를 포함할 수 있다. 숙주세포에 의해 인식되는 다양한 프로모터는 공지되어 있다. 원핵생물 숙주에 사용하기에 적합한 프로모터로 β락타마제 및 락토스 프로모터 시스템, 알칼리 포스타파제, 트립토판 프로모터 시스템, 하이브리드 프로모터, 예를 들어 tac 프로모터를 포함한다. 세균 시스템에서 사용되는 프로모터로는 SISP-1을 코딩하는 DNA에 작동가능하게 연결된 샤인-달가노(S.D.) 서열을 포함할 수 있다. 효모 숙주에 사용하기에 적합한 프로모터 서열로는 3-포스포글리세레이트 키나제 또는 다른 당 분해 효소들이 포함될 수 있다.The expression and cloning vectors may generally include a promoter operably linked to a nucleic acid sequence encoding wild-type CYP102A1, point mutants of wild-type CYP102A1, chimeric CYP102A1, and point mutants of chimeric CYP102A1 that induce mRNA synthesis. A variety of promoters recognized by host cells are known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan promoter systems, hybrid promoters such as the tac promoter. Promoters used in bacterial systems may include a Shine-Dalgarno (S.D.) sequence operably linked to DNA encoding SISP-1. Suitable promoter sequences for use in yeast hosts may include 3-phosphoglycerate kinase or other glycolytic enzymes.

본 발명의 다른 일 양태는 본 발명의 일 실시예에 따른 CYP102A1 돌연변이체들로 구성된 군으로부터 선택되는 하나 이상의 효소를 포함하는 3-히드록시 플로리진 제조용 조성물과 플로리진을 반응시키는 단계를 포함하는 3-히드록시 플로리진의 제조방법을 제공한다.Another aspect of the present invention is a composition for preparing 3-hydroxy phlorizin containing at least one enzyme selected from the group consisting of CYP102A1 mutants according to an embodiment of the present invention and 3 comprising the step of reacting phlorizin -Provides a method for producing hydroxy phlorizin.

본 발명의 일 실시예에 따르면, 상기 야생형 CYP102A1의 돌연변이체는 플로리진을 기질로 하여 3-히드록시 플로리진을 보다 효율적으로 생산할 수 있다.According to one embodiment of the present invention, the wild-type CYP102A1 mutant can more efficiently produce 3-hydroxy phlorizin using phlorizin as a substrate.

상기 야생형 CYP102A1의 돌연변이체와 플로리진의 반응 시간은, 예를 들어, 1분 내지 6시간, 예를 들어, 1시간 내지 4시간, 예를 들어, 3시간일 수 있으나, 이에 한정되는 것은 아니다. 상기 야생형 CYP102A1 및 이의 돌연변이체를 반응 기질인 플로리진과 반응 시 플로리진의 농도 및 효소의 농도는 적절하게 변경할 수 있다.The reaction time between the wild-type CYP102A1 mutant and phlorizin may be, for example, 1 minute to 6 hours, eg 1 hour to 4 hours, eg 3 hours, but is not limited thereto. When the wild-type CYP102A1 and its mutants are reacted with phlorizin as a reaction substrate, the concentration of phlorizin and the concentration of enzyme may be appropriately changed.

상기 제조 방법은 NADPH(환원된 니코틴아마이드 아데닌 디뉴클레오타이드 포스페이트, reduced nicotinamide adenine dinucleotide phosphate)-생성 시스템을 추가하는 단계를 더 포함할 수 있다. 상기 NADPH-생성 시스템은 본 발명이 속하는 분야에서 공지된 시스템, 예를 들어, 글루코스 6-포스페이트(glucose 6-phosphate), NADP-(니코틴아마이드 아데닌 디뉴클레오타이드 포스페이트) 및 효모 글루코스 6-포스페이트 디하이드로게나아제(glucose 6-phosphate dehydrogenase)를 사용할 수 있으나, 이에 한정되는 것은 아니다. 상기 NADPH-생성 시스템은 1μM 내지 1mM, 예를 들어, 100μM로 포함될 수 있다.The manufacturing method may further include adding a NADPH (reduced nicotinamide adenine dinucleotide phosphate)-generating system. The NADPH-producing system is a system known in the art, for example, glucose 6-phosphate, NADP- (nicotinamide adenine dinucleotide phosphate) and yeast glucose 6-phosphate dehydrogena Glucose 6-phosphate dehydrogenase may be used, but is not limited thereto. The NADPH-generating system may be included at 1 μM to 1 mM, for example, 100 μM.

본 발명의 다른 일 양태는 상기 3-히드록시 플로리진 제조용 조성물을 포함하는 3-히드록시 플로리진 생산용 키트를 제공한다.Another aspect of the present invention provides a kit for producing 3-hydroxy phlorizin comprising the composition for producing 3-hydroxy phlorizin.

상기 3-히드록시 플로리진 제조용 조성물은 야생형 CYP102A1의 돌연변이체로 구성된 군에서 선택되는 하나 이상의 효소를 포함할 수 있다.The composition for preparing 3-hydroxyphlorizin may include one or more enzymes selected from the group consisting of wild-type CYP102A1 mutants.

상기 야생형 CYP102A1의 돌연변이체는, 예를 들어, 서열번호 1로 표시되는 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/N213S/E267V, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R, D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V, R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R 및/또는 F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R일 수 있다.The wild-type CYP102A1 mutant is, for example, R47L/F81I/F87V/E143G/L188Q/N213S/E267V, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R of the wild-type CYP102A1 represented by SEQ ID NO: 1 , D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V, F42L/R47L/F81I/F87V/E143G/L188Q/E267V, T1S/R47L/F81I/F87V/E143G/T146A/L 188Q/E267V, R47L /F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R and/or F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R.

상기 야생형 CYP102A1의 돌연변이체는, 예를 들어, 서열번호 1로 표시되는 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/N213S/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F42L/R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E 및/또는 F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E로 구성된 군으로부터 선택되는 하나 이상일 수 있다.The wild-type CYP102A1 mutant is, for example, R47L/F81I/F87V/E143G/L188Q/N213S/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K81 of the wild-type CYP102A1 represented by SEQ ID NO: 1 3E /R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R/A4 74V/E558D/T664A/P675L/A678E /E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, D23G/R47L/F81I/F87V/F107L/D1 36G/E143G/L188Q/E267V/A474V /E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F42L /R47L/F81I/F87V/E143G/L188Q /E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y /Q1022E, T1S/R47L/F81I/F87V /E143G/T146A/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q 981R/A1008D/H1021Y/Q1022E, R47L /F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I88 1V/E887G/P894S/S954N/M967V /Q981R/A1008D/H1021Y/Q1022E and/or F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K 813E/R825S/R836H/E870N / I881V / E887G / P894S / S954N / M967V / Q981R / A1008D / H1021Y / Q1022E may be one or more selected from the group consisting of.

상기 3-히드록시 플로리진 생산용 키트는 NADPH-생성 시스템을 더 포함할 수 있다. 상기 NADPH-생성 시스템은 본 발명이 속하는 분야에서 공지된 시스템, 예를 들어, 글루코스 6-포스페이트(glucose 6-phosphate), NADP-(니코틴아마이드 아데닌 디뉴클레오타이드 포스페이트) 및 효모 글루코스 6-포스페이트 디하이드로게나아제(glucose 6-phosphate dehydrogenase)를 사용할 수 있으나, 이에 한정되는 것은 아니다.The kit for producing 3-hydroxyphlorizin may further include a NADPH-generating system. The NADPH-producing system is a system known in the art, for example, glucose 6-phosphate, NADP- (nicotinamide adenine dinucleotide phosphate) and yeast glucose 6-phosphate dehydrogena Glucose 6-phosphate dehydrogenase may be used, but is not limited thereto.

상기 3-히드록시 플로리진 생산용 키트는 그 외에 반응을 진행시키는데 필요한 시약을 추가로 포함할 수 있다.The kit for producing 3-hydroxyphlorizin may further include reagents necessary for proceeding with the reaction.

본 발명의 일 실시예에 따른 3-히드록시 플로리진 제조용 조성물, 키트, 및 이의 제조방법은 플로리진의 대사산물인 3-히드록시 플로리진을 경제적이면서 고효율로 대량생산할 수 있어, 기능성 바이오소재 및 약물의 개발 과정에서 3-히드록시 플로리진의 효능, 독성, 약물동력학 등을 평가하기 위하여 사용될 수 있으며, 기능성 소재개발의 리드 화합물이 될 수 있는 대사산물 유도체를 만드는데 사용될 수 있다.The composition, kit, and method for producing 3-hydroxy phlorizin according to an embodiment of the present invention can economically and efficiently mass-produce 3-hydroxy phlorizin, a metabolite of phlorizin, and thus functional biomaterials and drugs It can be used to evaluate the efficacy, toxicity, pharmacokinetics, etc. of 3-hydroxyphlorizin in the development process, and can be used to make metabolite derivatives that can be lead compounds for functional material development.

이하, 본 발명을 실시예에 의하여 상세히 설명한다. 그러나 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

<실시예><Example>

실시예 1. 점 돌연변이 유발에 의한 CYP102A1 돌연변이체의 구축Example 1. Construction of CYP102A1 mutants by point mutagenesis

야생형 CYP102A1의 점 돌연변이체(site-directed mutant) M16를 Kim 등이 제조한 방법과 같은 방법으로 제조하고(Kim DH, Ahn T, Jung HC, Pan JG, Yun CH. (2008) Generation of Human Metabolites of 7-Ethoxycoumarin by Bacterial Cytochrome P450 BM3. Drug Metabolism and Disposition 36(11):2166-2170. 논문의 2면 (page 2167) Materials and Methods의 construction of BM3 Mutants by Site-directed Mutagenesis 참조), M16(서열번호 2)으로 명명하였다(표 2).A site-directed mutant M16 of wild-type CYP102A1 was prepared in the same way as Kim et al. (Kim DH, Ahn T, Jung HC, Pan JG, Yun CH. (2008) Generation of Human Metabolites of 7-Ethoxycoumarin by Bacterial Cytochrome P450 BM3. 2) (Table 2).

BanHI/SacI 인식부위를 도입하기 위하여 하기 표 1에 기재된 프라이머 및 돌연변이를 일으키기 위한 PCR 프라이머(제노텍(XENOTECH), 한국)를 사용하였다. 아미노산 치환을 위한 코돈은 이탤릭체로 표시하였다. CYP102A1 돌연변이체를 코딩하는 유전자를 발현벡터 pCWori(Dr. F.W. Dahlquist, University of California, Santa Barbara, CA) 또는 pSE420(Invitrogen)로의 클로닝을 촉진할 수 있도록 설계된 프라이머를 사용하여 PCR 방법으로 pCWBM3로부터 증폭시켰다(Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnology and Bioengineering 111(7):1313- 1322. Page 1315 참조).In order to introduce the BanHI/SacI recognition site, PCR primers (XENOTECH, Korea) were used for primers and mutagenesis described in Table 1 below. Codons for amino acid substitutions are indicated in italics. The gene encoding the CYP102A1 mutant was amplified from pCWBM3 by PCR using primers designed to facilitate cloning into the expression vector pCWori (Dr. F.W. Dahlquist, University of California, Santa Barbara, CA) or pSE420 (Invitrogen). (Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs (see Biotechnology and Bioengineering 111(7):1313-1322. Page 1315).

하기 표 1에 기재된 프라이머를 사용하여 올리고뉴클레오티드 어셈블리를 실시하였다. 증폭된 유전자를 pCWBM BamHI/SacI 벡터의 BamHI/SacI 인식부위로 클로닝 하였다. 이러한 플라스미드로 Escherichia coli DH5α F'- IQ(인비트로겐)을 형질 전환시켰으며, 이는 CYP102A1 돌연변이 단백질을 발현시키기 위해서도 사용하였다. 돌연변이 유발 후, 목적하는 돌연변이가 일어났는지 여부를 DNA 시퀀싱을 의뢰하여(제노텍, 한국) 확인하였다.Oligonucleotide assembly was performed using the primers shown in Table 1 below. The amplified gene was cloned into the BamHI/SacI recognition site of the pCWBM BamHI/SacI vector. Escherichia coli DH5α F'-IQ (Invitrogen) was transformed with this plasmid, which was also used to express the CYP102A1 mutant protein. After mutagenesis, DNA sequencing was requested (Genotech, Korea) to confirm whether the desired mutation had occurred.

CYP102A1 돌연변이체의 구축을 위해 사용된 야생형 CYP102A1 아미노산 서열은 서열번호 1과 같고, 관례에 따라 첫번째 아미노산 메티오닌(M)은 아미노산 순서에 포함되지 않으며, 트레오닌(T)을 첫번째 아미노산으로 계산하였다.The wild-type CYP102A1 amino acid sequence used for construction of the CYP102A1 mutant is the same as SEQ ID NO: 1, and according to convention, the first amino acid methionine (M) is not included in the amino acid sequence, and threonine (T) is calculated as the first amino acid.

프라이머primer 서열order 서열번호sequence number BamHI forwardBamHI forward 5'-ATA GGA TCC ATG ACA ATT AAA GAA ATG CCT C-3'5'-ATA GGA TCC ATG ACA ATT AAA GAA ATG CCT C-3' 33 SacI reverseSacI reverse 5'-ATA GAG CTC GTA GTT TGT ATG ATC TTC AAA GTC AAA GTG-3'5'-ATA GAG CTC GTA GTT TGT ATG ATC TTC AAA GTC AAA GTG-3' 44 R47LR47L 5'- GCG CCT GGT CTG GTA ACG CG -3'5'-GCG CCT GGT CTG GTA ACG CG -3' 55 F81IF81I 5'- GTA CGT GAT ATT GCA GGA GAC -3'5'- GTA CGT GAT ATT GCA GGA GAC -3' 66 F87VF87V 5'- GAC GGG TTA GTG ACA AGC TGG -3'5'-GAC GGG TTA GTG ACA AGC TGG -3' 77 E143GE143G 5'- GAA GTA CCG GGC GAC ATG ACA -3'5'-GAA GTA CCG GGC GAC ATG ACA -3' 88 L188QL188Q 5'- ATG AAC AAG CAG CAG CGA GCA A -3'5'-ATG AAC AAG CAG CAG CGA GCA A -3' 99 E267VE267V 5'-T GCG GGA CAC GTG ACA ACA AGT -3'5'-T GCG GGA CAC GTG ACA ACA AGT -3' 1010

야생형의 CYP102A1의 점 돌연변이체Point mutants of wild-type CYP102A1 야생형 CYP102A1(서열번호 1)의 아미노산 치환 부위Amino acid substitution site of wild-type CYP102A1 (SEQ ID NO: 1) 참고문헌references M16M16 R47L/F81I/F87V/E143G/L188Q/E267VR47L/F81I/F87V/E143G/L188Q/E267V Kim DH et al., 2008Kim DH et al., 2008

*참고문헌**references*

: Kim DH, Kim KH, Kim DH, Liu KH, Jung HC, Pan JG, Yun CH. Generation of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3. Drug Metab Dispos 36:2166-2170, 2008: Kim DH, Kim KH, Kim DH, Liu KH, Jung HC, Pan JG, Yun CH. Generation of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3. Drug Metab Dispos 36:2166-2170, 2008

실시예 2. 키메라 CYP102A1의 구축Example 2. Construction of chimeric CYP102A1

야생형 CYP102A1의 점 돌연변이체의 헴 영역과 야생형 CYP102A1의 자연적 변형체(natural variant V2의 환원 효소 영역을 융합하여 선택적인 키메라 CYP102A1 단백질인 M16V2을 구축하였다. 야생형 CYP102A1의 점 돌연변이 체는 상기 실시예 1의 M16을 사용하였고, 자연적 변형체는 Bacillus megaterium 종의 자연적 변이체(표 3)을 사용하였다. A selective chimeric CYP102A1 protein, M16V2, was constructed by fusing the heme region of the point mutant of wild-type CYP102A1 with the reductase region of the natural variant V2 of wild-type CYP102A1. The point mutant of wild-type CYP102A1 was M16 of Example 1 was used, and natural variants of Bacillus megaterium species (Table 3) were used.

헴 영역과 환원효소 영역을 위하여 BamHI/SacI 및 SacI/XhoI을 사용하여 만들어진 키메라 CYP102A1 M16V2를 발현 벡터 pCW 벡터로 클로닝하였다. 이를 다시 돌연변이 시켜 키메라 CYP102A1 M16V2의 13종의 점 돌연변이체 B1, D8, M179, M221, M259, M328, M371, M375, M389, M524, M634, M788 및 M850을 제조하였다(Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnology and Bioengineering 111(7):1313- 1322. Page 1315 참조).The chimeric CYP102A1 M16V2 constructed using BamHI/SacI and SacI/XhoI for the heme region and reductase region was cloned into the expression vector pCW vector. 13 point mutants B1, D8, M179, M221, M259, M328, M371, M375, M389, M524, M634, M788 and M850 of chimera CYP102A1 M16V2 were prepared by mutating it again (Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.Biotechnology and Bioengineering 111(7):1313-1322. See Page 1315).

키메라 CYP102A1 및 이의 점 돌연변이체를 코딩하는 유전자를 발현벡터 pCWori(Dr. F.W. Dahlquist, University of California, Santa Barbara, CA) 또는 pSE420(인비트로겐)로의 클로닝을 촉진할 수 있도록 설계된 프라이머를 사용하여 PCR 방법으로 pCWBM3으로부터 증폭시켰다. 상기 표 1에 기재된 프라이머를 사용하여 올리고뉴클레오티드 어셈블리를 실시하였다. 증폭된 유전자를 pCWBM BamHI/SacI 벡터의 BamHI/SacI 인식부위로 클로닝 하였다. 이러한 플라스미드로 Escherichia coli DH5α F'-IQ(인비트로겐)을 형질 전환시켰으며, 이는 야생 형 CYP102A1의 돌연변이체 단백질을 발현시키기 위해서도 사용하였다. 돌연변이 유발 후, 목적하는 돌연변이가 일어났는지 여부를 DNA 시퀀싱을 의뢰하여(제노텍, 한국) 확인하였다.PCR method using primers designed to facilitate cloning of the gene encoding the chimeric CYP102A1 and its point mutants into the expression vector pCWori (Dr. F.W. Dahlquist, University of California, Santa Barbara, CA) or pSE420 (Invitrogen) amplified from pCWBM3. Oligonucleotide assembly was performed using the primers described in Table 1 above. The amplified gene was cloned into the BamHI/SacI recognition site of the pCWBM BamHI/SacI vector. Escherichia coli DH5α F'-IQ (Invitrogen) was transformed with these plasmids, which were also used to express mutant proteins of wild-type CYP102A1. After mutagenesis, DNA sequencing was requested (Genotech, Korea) to confirm whether the desired mutation had occurred.

균주strain 변종명Variant name 등록번호Registration Number Genomic DNAGenomic DNA 16S rRNA16S rRNA 16S-23S intergenic16S-23S intergenic KCCM 11745KCCM 11745 102A1.1102A1.1 (J04832)(J04832) FJ917385FJ917385 FJ969781FJ969781 IFO 12108IFO 12108 102A1.1102A1.1 (J04832)(J04832) FJ969756FJ969756 FJ969774FJ969774 ATCC 14581ATCC 14581 102A1.1102A1.1 (J04832)(J04832) FJ969751FJ969751 FJ969767FJ969767 KCCM 41415KCCM 41415 102A1.1102A1.1 (J04832)(J04832) FJ969762FJ969762 FJ969792FJ969792 KCTC 3712KCTC 3712 102A1.2102A1.2 FJ899078FJ899078 FJ969764FJ969764 FJ969795FJ969795 KCCM 12503KCCM 12503 102A1.3102A1.3 FJ899082FJ899082 FJ969761FJ969761 FJ969787FJ969787 ATCC 15451ATCC 15451 102A1.4102A1.4 FJ899085FJ899085 FJ969753FJ969753 FJ969768FJ969768 ATCC 10778ATCC 10778 102A1.5102A1.5 FJ899078FJ899078 FJ969746FJ969746 FJ969765FJ969765 KCCM 11938KCCM 11938 102A1.5102A1.5 FJ899078FJ899078 FJ969760FJ969760 FJ969786FJ969786 KCCM 11761KCCM 11761 102A1.5102A1.5 FJ899078FJ899078 FJ969757FJ969757 FJ969783FJ969783 KCCM 11776KCCM 11776 102A1.6102A1.6 FJ899081FJ899081 FJ969758FJ969758 FJ969784FJ969784 KCCM 11934KCCM 11934 102A1.6102A1.6 FJ899081FJ899081 FJ969759FJ969759 FJ969785FJ969785 ATCC 14945ATCC 14945 102A1.7102A1.7 FJ899084FJ899084 FJ969749FJ969749 FJ969766FJ969766 ATCC 21916ATCC 21916 102A1.8102A1.8 FJ899092FJ899092 FJ969755FJ969755 FJ969772FJ969772 KCTC 2194KCTC 2194 102A1.8102A1.8 FJ859036FJ859036 FJ969763FJ969763 FJ969794FJ969794 ATCC 19213ATCC 19213 102A1.9102A1.9 FJ899091FJ899091 FJ969754FJ969754 FJ969769FJ969769

* 참고문헌 *: Kang JY, Kim SY, Kim D, Kim DH, Shin SM, Park SH, Kim KH, Jung HC, Pan JG, Joung YH, Chi YT, Chae HZ, Ahn T, Yun CH (2011) Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium. AMB Express. 1(1):1.* References *: Kang JY, Kim SY, Kim D, Kim DH, Shin SM, Park SH, Kim KH, Jung HC, Pan JG, Joung YH, Chi YT, Chae HZ, Ahn T, Yun CH (2011) Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium . AMB Express. 1(1):1.

실시예 3. 야생형 CYP102A1 및 야생형 CYP102A1의 돌연변이체의 발현 및 정제Example 3. Expression and purification of wild-type CYP102A1 and mutants of wild-type CYP102A1

야생형 CYP102A1과 야생형 CYP102A1 돌연변이체의 유전자를 포함하는 플라스미드로 Escherichia coli DH5α F'-IQ을 형질전환 시켰다(Kim et al., Protein Expr Purif. 57:188-200., 2008). 하나의 콜로니로부터 적정량을 암피실린(100μg/mL)이 추가된 5mL Luria-Bertani 배지에 접종하여 37℃에서 배양하고, 이 배양물을 암피실린(100μg/mL)이 추가된 250mL Terrific Broth 배지에 접종하여 37℃에서 250rpm으로 흔들어 주며 OD600에서 0.8 정도가 될 때까지 배양하고, isopropyl-ß-D-thiogalactopyranoside(IPTG)를 최종 농도 0.5mM이 되도록 첨가하여 유전자 발현을 유도하였다. δ-aminolevulinic acid(1mM)을 첨가하였다. 발현이 유도된 후, 30℃에서 추가로 36 시간 더 배양하고 원심분리(15분, 5000g, 4℃)하여 세포를 수확하였다. 세포 펠렛을 TES 완충액(100mM Tris-HCl, pH7.6, 500mM sucrose, 0.5mM EDTA)에 재현탁시키고 초음파분해로 세포를 용해시켰다(sonicator; Misonix, Inc., Farmingdale, NY). 세포용해물을 100,000g, 90분, 4℃에서 원심분리한 후, 가용성 시토졸 분액(soluble cytosolic fraction)을 모아 활성을 측정하였다. 시토졸 분액을 50mM potassium phosphate 완충액(pH 7.4)에서 투석하여 -80℃에서 보관하였으며, 제조 후 1 개월 이내의 것을 실험에 사용하였다. Escherichia coli DH5α F'-IQ was transformed with plasmids containing wild-type CYP102A1 and wild-type CYP102A1 mutant genes (Kim et al., Protein Expr Purif. 57:188-200., 2008). An appropriate amount from one colony was inoculated into 5mL Luria-Bertani medium supplemented with ampicillin (100μg/mL) and cultured at 37°C, and the culture was inoculated into 250mL Terrific Broth medium supplemented with ampicillin (100μg/mL) and cultured at 37 °C. Shaking at 250 rpm at ℃ and incubated until the OD 600 is about 0.8, and isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce gene expression. δ-aminolevulinic acid (1 mM) was added. After expression was induced, the cells were harvested by incubation for an additional 36 hours at 30°C and centrifugation (15 minutes, 5000g, 4°C). Cell pellets were resuspended in TES buffer (100 mM Tris-HCl, pH7.6, 500 mM sucrose, 0.5 mM EDTA) and cells were lysed by sonication (sonicator; Misonix, Inc., Farmingdale, NY). After the cell lysate was centrifuged at 100,000g, 90 minutes, 4° C., the soluble cytosolic fraction was collected and the activity was measured. The cytosolic fraction was dialyzed in 50mM potassium phosphate buffer (pH 7.4) and stored at -80°C, and was used within 1 month after preparation for the experiment.

CYP102A1의 농도는 CO-difference 스펙트럼으로부터 결정하였으며, 이때 사용한 ε은 91mM/cm이었다(Omura and Sato (1964) THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem 239:2379-2385). 야생형 및 돌연변이체 모두 통상적으로 300-700nM P450을 얻을 수 있었다. 야생형 CYP102A1 및 야생형 CYP102A1의 돌연변이체의 발현정도는 1.0~2.0nmol P450/mg cytosolic protein의 범위였다. 제조된 야생형 CYP102A1의 돌연변이체들 중에서 사람에 있어서의 기질에 대한 촉매 활성이 큰 키메라 CYP102A1의 돌연변이체인 M16V2 및 이의 돌연변이체 B1, D8, M179, M221, M259, M328, M371, M375, M389, M524, M634, M788 및 M850을 선택하였고, 하기 표 4에 야생형 CYP102A1의 돌연변이체에서의 아미노산이 치환된 부위를 나타내었다(Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnology and Bioengineering 111(7):1313-1322).The concentration of CYP102A1 was determined from the CO-difference spectrum, where ε was 91 mM/cm (Omura and Sato (1964) THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem 239 :2379-2385). Both wild-type and mutants could typically obtain 300-700 nM P450. The expression levels of wild-type CYP102A1 and wild-type CYP102A1 mutants ranged from 1.0 to 2.0 nmol P450/mg cytosolic protein. Among the prepared wild-type CYP102A1 mutants, M16V2, a mutant of chimeric CYP102A1 with high catalytic activity for human substrates, and its mutants B1, D8, M179, M221, M259, M328, M371, M375, M389, M524, M634, M788 and M850 were selected, and Table 4 below shows amino acid substitution sites in wild-type CYP102A1 mutants (Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.Biotechnology and Bioengineering 111(7):1313-1322).

키메라 CYP102A1 및 이의 점 돌연변이체Chimeric CYP102A1 and point mutants thereof 야생형 CYP102A1(서열번호 1)의 아미노산 치환 부위Amino acid substitution site of wild-type CYP102A1 (SEQ ID NO: 1) 서열번호sequence number M16V2M16V2 R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E R47L/F81I/F87V/E143G/L188Q/E267V/ A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N /M967V/Q981R/A1008D/H1021Y/ Q1022E 1111 B1B1 R47L/F81I/F87V/E143G/L188Q/E267V/D351GR47L/F81I/F87V/E143G/L188Q/E267V/D351G 1212 D8D8 R47L/F81I/F87V/E143G/L188Q/E267V/Y334C/A335V/D369GR47L/F81I/F87V/E143G/L188Q/E267V/Y334C/A335V/D369G 1313 M179M179 R47L/F81I/F87V/E143G/L188Q/N213S/E267VR47L/F81I/F87V/E143G/L188Q/N213S/E267V 1414 M221M221 F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408RF11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R 1515 M259M259 R47L/F81I/F87V/E143G/T149S/L188Q/E267V/S270GR47L/F81I/F87V/E143G/T149S/L188Q/E267V/S270G 1616 M328M328 R47L/F81I/F87V/E143G/K187E/L188Q/V211M/E267A/S274TR47L/F81I/F87V/E143G/K187E/L188Q/V211M/E267A/S274T 1717 M371M371 D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267VD23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V 1818 M375M375 R47L/F81I/F87V/S106C/Q109R/E143G/L188Q/E267V/D338ER47L/F81I/F87V/S106C/Q109R/E143G/L188Q/E267V/D338E 1919 M389M389 D23G/R47L/F81I/D84N/F87V/E143G/G154S/M185V/L188Q/E267VD23G/R47L/F81I/D84N/F87V/E143G/G154S/M185V/L188Q/E267V 2020 M524M524 F42L/R47L/F81I/F87V/E143G/L188Q/E267VF42L/R47L/F81I/F87V/E143G/L188Q/E267V 2121 M634M634 T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267VT1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V 2222 M788M788 R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383RR47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R 2323 M850M850 F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408RF11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R 2424

* 상기 돌연변이체에 있어 밑줄 친 부분은 야생형 CYP102A1의 환원효소 영역(reductase domain)을 돌연변이 시킴.* In the above mutant, the underlined part mutates the reductase domain of wild-type CYP102A1.

*참고문헌**references*

: Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH, Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol. Bioeng. 111:1313-1322, 2014;: Kang JY, Ryu SH, Park SH, Cha GS, Kim DH, Kim KH, Hong AW, Ahn T, Pan JG, Joung YH, Kang HS, Yun CH, Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol. Bioeng. 111:1313-1322, 2014;

: Le TK, Jang HH, Nguyen HT, Doan TT, Lee GY, Park KD, Ahn T, Joung YH, Kang HS, Yun CH, Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3. Enzyme Microb Technol. 97:34-42, 2017.: Le TK, Jang HH, Nguyen HT, Doan TT, Lee GY, Park KD, Ahn T, Joung YH, Kang HS, Yun CH, Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450 BM3. Enzyme Microb Technol. 97:34-42, 2017.

실험예 1. CYP102A1 돌연변이체에 의한 플로리진의 대사산물 생성의 확인Experimental Example 1. Confirmation of production of metabolites of phlorizin by CYP102A1 mutants

야생형 CYP102A1(BM3 wt) 및 이의 돌연변이체들이 플로리진을 산화시켜 3-히드록시화 할 수 있는지 확인하였다. 구체적으로, 100mM 인산칼륨 완충액(pH 7.4) 0.25mL에 각각 야생형 CYP102A1 및 이의 돌연변이체들 0.4μM, 플로리진(0.2mM)을 넣어 전형적인 정상 상태(steady-state) 반응을 수행하였다. It was confirmed whether wild-type CYP102A1 (BM3 wt) and its mutants could oxidize phlorizin to 3-hydroxylate it. Specifically, a typical steady-state reaction was performed by adding 0.4 μM of wild-type CYP102A1 and its mutants and phlorizin (0.2 mM) to 0.25 mL of 100 mM potassium phosphate buffer (pH 7.4).

반응을 시작하기 위하여 NADPH-생성 시스템(최종 농도: 1mL 당 10mM glucose 6-phosphate, 0.5mM NADP+ 및 1 IU yeast glucose 6-phosphate dehydrogenase)을 첨가하였다. 반응액을 37℃에서 60분간 반응시키고, 얼음으로 빙냉시킨 에틸아세테이트 600μL로 반응을 종결시켰다. A NADPH-generating system (final concentrations: 10 mM glucose 6-phosphate, 0.5 mM NADP + and 1 IU yeast glucose 6-phosphate dehydrogenase per 1 mL) was added to initiate the reaction. The reaction solution was reacted at 37° C. for 60 minutes, and the reaction was terminated with 600 μL of ice-cooled ethyl acetate.

실험예 2. HPLC 분석Experimental Example 2. HPLC analysis

상기 실험예 1의 반응혼합물을 원심분리한 후, 유기용매 층 180μL를 분리하여 질소가스 하에서 증발시키고 HPLC로 분석하였다. 구체적으로, 시료를 Gemini C18 컬럼(4.6x150mm, 5μm, 110Å; phenomenex, Torrance, CA)에 주입하였다. 이동상 A는 0.5% 메탄올과 0.1% 포름산을 포함하는 증류수, 이동상 B는 아세토니트릴을 사용하였다. 생성물 형성은 A285에서 분석되었으며, 이동상 A/B(v/v)를 그래디언트 펌프(LC-20AD, Shimadzu, Kyoto, Japan)를 사용하여 1.0mL/min의 속도로 흘려주었다. 그래디언트 조건은 다음과 같았다: 0~3 분 사이 B이동상 9%; 3~20분 이동상 30%; 20~21분 9% 이동상 B; 21~30분 B 이동상 9% 에서 제어.After the reaction mixture of Experimental Example 1 was centrifuged, 180 μL of the organic solvent layer was separated, evaporated under nitrogen gas, and analyzed by HPLC. Specifically, samples were injected onto a Gemini C18 column (4.6x150 mm, 5 μm, 110 Å; phenomenex, Torrance, CA). Mobile phase A was distilled water containing 0.5% methanol and 0.1% formic acid, and mobile phase B was acetonitrile. Product formation was analyzed in A 285 , and mobile phase A/B (v/v) was flowed at a rate of 1.0 mL/min using a gradient pump (LC-20AD, Shimadzu, Kyoto, Japan). The gradient conditions were as follows: 9% mobile phase B between 0 and 3 min; 3-20 minutes 30% mobile phase; 20-21 min 9% mobile phase B; 21-30 minutes Control at 9% B mobile phase.

CYP102A1 돌연변이체에 의한 플로리진 3-히드록실화의 운동 매개 변수를 결정하기 위해 0.01-1mM 플로리진, 0.40μM 효소 및 NADPH 생성 시스템을 사용하였다. 37℃에서 60분 동안 배양한 후, HPLC를 사용하여 생성물의 형성 속도를 결정하였다. 기질 용액은 에탄올에서 준비하고 효소 반응에서 최종 유기 용매 농도 <1 % (v/v)로 희석하였다. 결과는 GraphPad Prism (GraphPad Software, San Diego, CA, USA)을 사용한 비선형 회귀 분석을 사용하여 결정하였다. 데이터는 표준 Michaelis-Menten 방정식을 통해 도출하였다.To determine the kinetic parameters of phlorizin 3-hydroxylation by CYP102A1 mutants, 0.01-1 mM phlorizin, 0.40 μM enzyme and a NADPH generating system were used. After incubation at 37° C. for 60 min, the rate of product formation was determined using HPLC. Substrate solutions were prepared in ethanol and diluted to a final organic solvent concentration <1% (v/v) in the enzymatic reaction. Results were determined using non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data were derived through the standard Michaelis-Menten equation.

플로리진 히드록실화의 시간 경과 분석은 100mM 인산 칼륨 완충액 (pH 7.4) 및 0.40μM 효소에 대하여 200μM 플로리진으로 측정되었다. 반응은 NADPH 생성 시스템을 추가하고 37℃에서 30분, 60분, 90분, 2시간 및 3시간 동안 배양하여 시작되었다. 생성물 형성은 전술한 바와 같이 HPLC를 사용하여 분석하였다.Time course analysis of phlorizin hydroxylation was measured with 200 μM phlorizin in 100 mM potassium phosphate buffer (pH 7.4) and 0.40 μM enzyme. The reaction was started by adding the NADPH generating system and incubating at 37°C for 30 minutes, 60 minutes, 90 minutes, 2 hours and 3 hours. Product formation was analyzed using HPLC as described above.

실험예 3. LC-MS 분석Experimental Example 3. LC-MS analysis

야생형 CYP102A1의 돌연변이체로부터 생산된 플로리진의 대사산물을 동정하기 위하여, 플로리진 및 이의 대사산물의 LC 프로파일과 단편(fragmentation) 패턴을 비교하여 LC-MS 분석을 수행하였다.In order to identify metabolites of phlorizin produced from wild-type CYP102A1 mutants, LC-MS analysis was performed by comparing LC profiles and fragmentation patterns of phlorizin and its metabolites.

반응 잔류물을 100μL 이동상에 볼텍스 믹싱으로 재구성시키고, 제조된 용액의 적정량(5μL)을 LC 컬럼에 주입하였다. LC-MS분석은 HESI II프로브(thermo Fisher Scientific, USA)와 함께 가열된 전기 분무 이온화 인터페이스가 있는 Thermo Scientific AccelaTM, XcaliburTM 3.0 및 TSQ QuantumTM Access MAX 시스템에서 수행하였다. 분리는 ZorBax SB-C18(4.6x250mm, 5μM, 80Å; Agilent Technologies, USA); 이동상 A는 0.5%(v/v) 메탄올, 0.1% 포름산(v/v)을 포함하는 증류수, 이동상 B는 아세토니트릴을 사용하여 1.0mL/min의 속도로 흘려주었다. The reaction residue was reconstituted by vortex mixing in 100 μL mobile phase, and an appropriate amount (5 μL) of the prepared solution was injected into the LC column. LC-MS analysis was performed on a Thermo Scientific Accela™, Xcalibur™ 3.0 and TSQ Quantum™ Access MAX system with a heated electrospray ionization interface with a HESI II probe (thermo Fisher Scientific, USA). Separation was performed using ZorBax SB-C18 (4.6x250 mm, 5 μM, 80 Å; Agilent Technologies, USA); Mobile phase A was distilled water containing 0.5% (v/v) methanol and 0.1% formic acid (v/v), and mobile phase B was flowed at a rate of 1.0 mL/min using acetonitrile.

이동상 B의 초기 조성은 9%, 11분후 이동상 B 조성은 17분에 걸쳐 15%로 증가하고, 7분에 걸쳐 17%로 증가하고, 17분에 걸쳐 60%로 증가하고, 1분에 걸쳐 100%로 증가하고 마지막으로 10분에 걸쳐 초기 조건으로 재 평형화 하였다. 컬럼과 자동 시료 주입기의 온도는 각각 30℃와 4℃로 유지되었다. 질량 스펙트럼은 플로리진의 대사 산물을 확인하기 위하여 네거티브 모드에서 전지 분무 이온화에 의해 기록되었고, 전체 스캔 질량 분석법이 사용되었다. 충돌 에너지와 스캔 속도는 각각 20V 및 0.5spectral/s였다. 기화기 온도와 모세관 온도는 모두 320℃ 였고, 질소 차단 가스 압력 및 보조 가스 압력은 각각 60psi 및 20psi였으며 분무 전압은 3000V로 진행되었다.The initial composition of mobile phase B is 9%, after 11 minutes the mobile phase B composition increases to 15% over 17 minutes, to 17% over 7 minutes, to 60% over 17 minutes, and to 100% over 1 minute. % and finally re-equilibrated to initial conditions over 10 minutes. The temperature of the column and autosampler was maintained at 30 °C and 4 °C, respectively. Mass spectra were recorded by electrospray ionization in negative mode and full scan mass spectrometry was used to identify metabolites of phlorizin. The collision energy and scan rate were 20 V and 0.5 spectral/s, respectively. The vaporizer temperature and capillary temperature were both 320° C., the nitrogen barrier gas pressure and auxiliary gas pressure were 60 psi and 20 psi, respectively, and the spraying voltage was 3000 V.

실험결과Experiment result

1. CYP102A1 돌연변이체에 의한 플로리진의 히드록실화1. Hydroxylation of phlorizin by CYP102A1 mutants

CYP102A1이 플로리진을 히드록실화하는 능력을 결정하기 위해 폴리다틴(polydatin), 나린진 DC(naringin DC) 및 플로레틴(phloretin)과 같은 폴리페놀 화합물의 높은 히드록실화 활성을 갖는 WT 및 돌연변이체 세트의 촉매 활성을 결정하기 위해, 37℃에서 30분 동안 200μM 플로리진에 대하여 실험을 진행하였다(도 1). A set of WT and mutants with high hydroxylation activity of polyphenolic compounds such as polydatin, naringin DC and phloretin to determine the ability of CYP102A1 to hydroxylate phloregin In order to determine the catalytic activity of , the experiment was conducted for 200 μM phlorizin at 37 ° C. for 30 minutes ( FIG. 1 ).

선택은 돌연변이체의 발현 수준과 플로리진 히드록실화의 촉매 활성을 기반으로 하였다. 야생형 CYP102A1의 돌연변이체 중 M179, M221, M259, M371, M375, M524, M634, M788 및 M850이 M16V2(0.03min-1)보다 높은 촉매 활성을 나타내고, 특히 M221, M371 및 M850은 M16V2에 비하여 현저히 높은 촉매 활성을 나타내고 있음을 알 수 있다(도 1). 또한 주요 산물을 만드는 돌연변이 M371과 M850의 활성은 M16V2보다 100배 더 높았다.Selection was based on the expression level of the mutants and the catalytic activity of phlorizin hydroxylation. Among the wild-type CYP102A1 mutants, M179, M221, M259, M371, M375, M524, M634, M788 and M850 exhibit higher catalytic activity than M16V2 (0.03 min -1 ), and in particular, M221, M371 and M850 exhibit significantly higher catalytic activity than M16V2. It can be seen that it exhibits catalytic activity (FIG. 1). In addition, the activities of the mutations M371 and M850, which make the major products, were 100 times higher than those of M16V2.

주요 대사 산물과 기질의 정체는 HPLC(도 2)와 LC-MS(도 3)를 비교하여 확인되었다. M371에 의한 플로리진 산물을 확인하기 위해 LC-MS 분석을 네거티브 모드로 수행하였다(도 3, NADPH 없는 경우 (A); NADPH 있는 경우 (B); 플로리진 (C)의 양성자화된 분자 이온 ([M-H]-) (C); 주요 반응물(M1) (D)). 플로리진([M-H]-)은 m/z 435이고 주요 반응물(M1)은 m/z 451임을 확인하였다. 이 결과는 M371이 단 하나의 주요 반응물인 모노히드록실화 반응물만 생산함을 나타낸다.The identities of major metabolites and substrates were confirmed by comparing HPLC (Fig. 2) and LC-MS (Fig. 3). LC-MS analysis was performed in negative mode to confirm the phlorizin product by M371 (Fig. 3, without NADPH (A); with NADPH (B); protonated molecular ion of phlorizin (C) ( [MH] - ) (C);Main reactant (M1) (D)). It was confirmed that phlorizin ([MH] - ) was m/z 435 and the main reactant (M1) was m/z 451. These results indicate that M371 produces only one major reactant, the monohydroxylation reactant.

2. CYP102A1 및 β-Glucosidase에 의한 플로리진의 반응물의 특징2. Characteristics of phlorizin reaction by CYP102A1 and β-Glucosidase

플로리진 200μM을 NADPH 생성 시스템의 존재 하에 M371 0.4μM과 함께 배양하여 주요 대사 산물의 형성을 확인하였다(도 4, NADPH 없는 경우 (a); NADPH 있는 경우 (b); 반응 종료 후 β-글루코시다아제 첨가 (c); 3-OH 플로레틴 (tR = 32.4분), 플로레틴 (tR = 34.6분) 및 플로리진 (tR = 30.1분)의 표준 화합물 (d) 각 화합물의 농도는 10μM). 2.4 배 부피의 빙냉 에틸 아세테이트로 반응을 중단한 후 기질과 대사 산물을 추출하고 건조시켰다. 이어서 12시간 동안 플로리진의 반응 혼합물 (Vt = 0.25mL)에 β-글루코시다아제 (5U)를 첨가하여 β-글루코시다아제의 처리에 따른 M1과 기질의 생성물 프로파일의 변화를 확인하였다. 200 μM of phlorizin was incubated with 0.4 μM of M371 in the presence of a NADPH generating system to confirm the formation of major metabolites (FIG. 4, without NADPH (a); with NADPH (b); after completion of the reaction, β-glucosida Aze addition (c) Standard compounds of 3-OH phloretin (t R = 32.4 min), phloretin (t R = 34.6 min) and phlorizin (t R = 30.1 min) (d) The concentration of each compound is 10 μM ). After stopping the reaction with 2.4 volumes of ice-cold ethyl acetate, substrates and metabolites were extracted and dried. Subsequently, β-glucosidase (5U) was added to the phlorizin reaction mixture (V t = 0.25 mL) for 12 hours, and changes in product profiles of M1 and the substrate according to the treatment with β-glucosidase were confirmed.

M1 산물과 플로리진은 각각 3-OH 플로레틴과 플로레틴으로 전환되었으며(도 4, c 행) 일부 플로리진은 여전히 남아 있음을 확인하였다. 이는 CYP102A1의 처리에 의해 플로리진으로부터 3-OH 플로리진이 생성됨을 나타낸다.It was confirmed that the M1 product and phlorizin were converted to 3-OH phloretin and phloretin, respectively (Fig. 4, row c), and some phlorizin remained. This indicates that 3-OH phlorizin is produced from phlorizin by treatment of CYP102A1.

모노 히드록실화 생성물이 3-OH 플로리진인지 확인하기 위해, M371 처리된 플로리진 및 후속 β-글루코시다아제 처리의 반응 혼합물로 LC-MS/MS 분석을 수행하였다(도 5). M371로 처리한 플로리진의 크로마토그램(TIC)과 β-글루코시다아제의 후속 처리는 플로리진(기질), 대사 산물 M2 및 대사 산물 M(플로레틴)의 세 가지 피크를 보여준다(도 5A). 또한, MS 스펙트럼은 추가 단편화(MS2)가 있는 플로레틴 (M) 및 딸 이온 (M2)의 양성자화된 분자 이온을 보여준다(도 5B 및 C). 또한, MS 스펙트럼은 대사 산물(M2)의 양성자 분자 이온과 추가 단편화(도 5D 및 E)와 함께 그 딸 이온(MS2)을 보여준다. 이러한 결과는 대사 산물 M2가 초기 히드록실화 및 플로리진의 후속 탈당화 반응을 통해 형성됨을 나타낸다.To confirm that the monohydroxylation product was 3-OH phlorizin, LC-MS/MS analysis was performed on the reaction mixture of M371 treated phlorizin followed by β-glucosidase treatment (FIG. 5). Chromatogram (TIC) of phlorizin treated with M371 and subsequent treatment with β-glucosidase showed three peaks: phlorizin (substrate), metabolite M2 and metabolite M (phloretin) (Fig. 5A). MS spectra also show protonated molecular ions of phloretin (M) and daughter ion (M2) with additional fragmentation (MS2) (Fig. 5B and C). The MS spectrum also shows the proton molecular ion of metabolite (M2) and its daughter ion (MS2) with further fragmentation (Fig. 5D and E). These results indicate that metabolite M2 is formed through initial hydroxylation and subsequent deglycosylation of phlorizin.

상기 M2가 3-OH 플로레틴이라는 것을 증명하기 위해 CYP102A1 M10 돌연변이를 통해 플로레틴에서 형성된 3-OH 플로레틴(도 7)의 단편화 패턴(도 5E)을 비교하였다(Nguyen, N.A.; Jang, J.; Le, T.-K.; Nguyen, T.H.H.; Woo, S.-M.; Yoo, S.-K.; Lee, Y.J.; Park, K.D.; Yeom, S.-J.; Kim, G.-J.; et al. Biocatalytic Production of a Potent Inhibitor of Adipocyte Differentiation from Phloretin Using Engineered CYP102A1. J. Agric. Food Chem. 2020, 68, 6683-6691). 그 결과 대사체의 양성자화 된 분자 이온과 추가적인 단편화가 있는 딸 이온을 보여주는 두 샘플의 MS 스펙트럼이 정확히 일치하였다. 이를 통해 M2가 초기 히드록실화 및 이후의 플로리진 탈당화 반응을 통해 형성된 3-OH 플로레틴임을 알 수 있다.In order to prove that the M2 is 3-OH phloretin, the fragmentation pattern (Fig. 5E) of 3-OH phloretin (Fig. 7) formed from phloretin through the CYP102A1 M10 mutation was compared (Nguyen, N.A.; Jang, J. ;Le, T.-K.; Nguyen, T.H.H.; Woo, S.-M.; Yoo, S.-K.; Lee, Y.J.; Park, K.D.; Yeom, S.-J.; Kim, G.- J.; et al. Biocatalytic Production of a Potent Inhibitor of Adipocyte Differentiation from Phloretin Using Engineered CYP102A1. J. Agric. Food Chem. 2020, 68, 6683-6691). As a result, the MS spectra of the two samples, showing the protonated molecular ion of the metabolite and the daughter ion with additional fragmentation, matched exactly. From this, it can be seen that M2 is 3-OH phloretin formed through initial hydroxylation and subsequent deglycosylation of phlorizin.

3. CYP102A1 돌연변이체에 의한 플로리진 히드록실화의 운동 파라미터 및 TTN3. Kinetic parameters and TTN of phlorizin hydroxylation by CYP102A1 mutants

4개의 돌연변이체를 추가 실험을 위해 선택하여 플로리진 3-히드록실화의 운동 매개 변수 및 총 회전율(TTN, turnover number)을 결정하였다. 3 개의 돌연변이(M221, M371, M850)는 효소 라이브러리를 만들기 위한 주형으로 사용된 M16V2보다 높은 활성을 가졌다.Four mutants were selected for further experiments to determine the kinetic parameters and total turnover number (TTN) of phlorizin 3-hydroxylation. Three mutants (M221, M371, M850) had higher activity than M16V2 used as a template for constructing the enzyme library.

하기 표 5은 돌연변이 M221, M371, M850 및 M16V2에 의한 플로리진으로부터의 히드록실화 생성물 형성의 정상 상태 역학을 보여준다. M221은 18min-1의 가장 높은 kcat 값을 가지며 이는 M16V2 (0.16min-1)에 비하여 112배 높은 수치를 나타낸다. 또한 M371 및 M850은 kcat 값이 각각 88배, 39배 증가하였다. 상기 M221, M371 및 M850 돌연변이체는 Km 값이 2.8~5.8 배 증가한 것으로 나타났다. 또한 M221 및 M371의 촉매 효율은 M16V2에 비해 20 ~ 25 배 증가하였다.Table 5 below shows the steady state kinetics of hydroxylation product formation from phlorizin by the mutations M221, M371, M850 and M16V2. M221 has the highest k cat value of 18min -1 , which is 112 times higher than that of M16V2 (0.16min -1 ). In addition, M371 and M850 increased k cat values by 88 and 39 times, respectively. The M221, M371 and M850 mutants showed a 2.8-5.8 fold increase in K m value. In addition, the catalytic efficiency of M221 and M371 increased 20 to 25 times compared to M16V2.

효소enzyme k cat (min-1 ) k cat (min -1 ) K m (μM) K m (μM) k cat /K m (min-1μM-1) k cat /K m (min -1 µM -1 ) M221M221 18 ± 418±4 1540 ± 5001540±500 0.012 ± 0.0040.012 ± 0.004 M371M371 14 ± 314±3 979 ± 315979 ± 315 0.015 ± 0.0050.015 ± 0.005 M850M850 6.3 ± 0.56.3 ± 0.5 747 ± 497747 ± 497 0.00084 ± 0.000070.00084 ± 0.00007 M16V2M16V2 0.16 ± 0.010.16 ± 0.01 267 ± 34267 ± 34 0.00059 ± 0.000080.00059 ± 0.00008

또한, 돌연변이체 M221, M371 및 M16V2를 사용하여 플로리진 히드록실화의 TTN을 측정하였다. 30분, 60분, 90분, 120분, 150분, 180분 동안 0.2mM 플로리진으로 생성물 형성을 위한 상기 돌연변이체의 TTN(nmol product/nmol P450)을 측정했을 때 전체 범위는 3 ~ 198 TTN이었다. M371은 최대 60분의 배양 시간으로 대사산물 형성을 증가시켰고 안정기에 도달하였다. 또한 M221과 M371에 의한 3-OH 플로리진 생산은 60분에 최대에 도달한 것으로 나타났다(도 7).In addition, TTN of phlorizin hydroxylation was determined using mutants M221, M371 and M16V2. When the TTN (nmol product/nmol P450) of the mutant for product formation was measured with 0.2 mM phlorizin for 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes, the overall range was 3 to 198 TTN. was M371 increased metabolite formation and reached a plateau with incubation times up to 60 minutes. In addition, the production of 3-OH phlorizin by M221 and M371 was found to reach a maximum at 60 minutes (FIG. 7).

4. CYP102A1 돌연변이체에 대한 플로리진의 스펙트럼 적정4. Spectral Titration of Phlorizin to CYP102A1 Mutants

플로리진이 CYP102A1에 결합할 수 있는지 확인하기 위해, 플로리진을 사용하여 스펙트럼 결합 적정을 수행하였다(도 8). 플로리진은 415nm에서 피크를, 392nm에서 최저점을 나타냈는데, 이는 리간드의 유형 II 결합과 일치하며, 이는 P450 활성 부위에서 물 분자가 방출되고 리간드의 염기성 질소 원자가 헴 철에 배위됨을 나타냅니다. 증가하는 플로리진 농도의 존재는 더 낮은 플로리진 농도에서 ΔA를 향상시키고 더 낮은 파장으로 저점 위치에서 연속적인 이동을 일으켰다. 모든 시험 된 돌연변이체는 6.8~9.4μM 범위에서 플로리진에 대한 높은 결합 친화성을 나타냈다. 특히 M620은 Kd 값 6.8μM의 가장 높은 친화도를 가진 플로리진에 결합되었음을 알 수 있다.To confirm whether phlorizin can bind to CYP102A1, spectral binding titration was performed using phlorizin (FIG. 8). Phlorizin exhibited a peak at 415 nm and a trough at 392 nm, consistent with type II binding of the ligand, indicating release of water molecules from the P450 active site and coordination of the ligand's basic nitrogen atom to heme iron. The presence of increasing phlorizin concentrations enhanced ΔA at lower phlorizin concentrations and caused a continuous shift in the trough position to lower wavelengths. All tested mutants showed high binding affinity to phlorizin in the range of 6.8 to 9.4 μM. In particular, it can be seen that M620 was bound to phlorizin with the highest affinity with a K d value of 6.8 μM.

전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

본 발명의 범위는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> COMPOSITION FOR PREPARING 3-HYDROXY PHLORIZIN <130> P-210179 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> Bacillus megaterium CYP102A1 (wild type) <400> 1 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 2 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M16 <400> 2 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> BamHI forward primer <400> 3 ataggatcca tgacaattaa agaaatgcct c 31 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> SacI reverse primer <400> 4 atagagctcg tagtttgtat gatcttcaaa gtcaaagtg 39 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R47L primer <400> 5 gcgcctggtc tggtaacgcg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F81I primer <400> 6 gtacgtgata ttgcaggaga c 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F87V primer <400> 7 gacgggttag tgacaagctg g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> E143G primer <400> 8 gaagtaccgg gcgacatgac a 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> L188Q primer <400> 9 atgaacaagc agcagcgagc aa 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> E267V primer <400> 10 tgcgggacac gtgacaacaa gt 22 <210> 11 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M16V2 <400> 11 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 12 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant B1 <400> 12 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Gly Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 13 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant D8 <400> 13 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Cys Val Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Gly Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 14 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M179 <400> 14 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Ser Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 15 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M221 <400> 15 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Tyr Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu Arg Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 16 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M259 <400> 16 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Ser Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Gly Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 17 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M328 <400> 17 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Glu Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Met Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Ala Thr Thr Ser Gly Leu 260 265 270 Leu Thr Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 18 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M371 <400> 18 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Gly Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Leu Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Gly Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 19 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M375 <400> 19 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Cys Phe Ser Arg Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Glu Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 20 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M389 <400> 20 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Gly Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asn Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Ser Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Val Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 21 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M524 <400> 21 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Leu Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 22 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M634 <400> 22 Ser Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Ala Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 23 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M788 <400> 23 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Asn Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asp Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Ile Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Arg Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 24 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M850 <400> 24 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Tyr Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Gly Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu Arg Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> COMPOSITION FOR PREPARING 3-HYDROXY PHLORIZIN <130> P-210179 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 1048 <212> PRT <213> artificial sequence <220> <223> Bacillus megaterium CYP102A1 (wild type) <400> 1 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 2 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M16 <400> 2 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 3 <211> 31 <212> DNA <213> artificial sequence <220> <223> BamHI forward primer <400> 3 ataggatcca tgacaattaa agaaatgcct c 31 <210> 4 <211> 39 <212> DNA <213> artificial sequence <220> <223> SacI reverse primer <400> 4 atagagctcg tagttgtat gatcttcaaa gtcaaagtg 39 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> R47L primer <400> 5 gcgcctggtc tggtaacgcg 20 <210> 6 <211> 21 <212> DNA <213> artificial sequence <220> <223> F81I primer <400> 6 gtacgtgata ttgcaggaga c 21 <210> 7 <211> 21 <212> DNA <213> artificial sequence <220> <223> F87V primer <400> 7 gacgggttag tgacaagctg g 21 <210> 8 <211> 21 <212> DNA <213> artificial sequence <220> <223> E143G primer <400> 8 gaagtaccgg gcgacatgac a 21 <210> 9 <211> 22 <212> DNA <213> artificial sequence <220> <223> L188Q primer <400> 9 atgaacaagc agcagcgagc aa 22 <210> 10 <211> 22 <212> DNA <213> artificial sequence <220> <223> E267V primer <400> 10 tgcgggacac gtgacaacaa gt 22 <210> 11 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M16V2 <400> 11 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 12 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant B1 <400> 12 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Gly Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 13 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant D8 <400> 13 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Cys Val Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Gly Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 14 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M179 <400> 14 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Ser Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 15 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M221 <400> 15 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Tyr Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu Arg Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 16 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M259 <400> 16 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Ser Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Gly Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 17 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M328 <400> 17 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Glu Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Met Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Ala Thr Thr Ser Gly Leu 260 265 270 Leu Thr Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 18 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M371 <400> 18 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Gly Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Leu Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Gly Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 19 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M375 <400> 19 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Cys Phe Ser Arg Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Glu Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 20 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M389 <400> 20 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Gly Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asn Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Ser Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Val Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 21 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M524 <400> 21 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Leu Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 22 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M634 <400> 22 Ser Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Ala Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 23 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M788 <400> 23 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Asn Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asp Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Ile Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Arg Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045 <210> 24 <211> 1048 <212> PRT <213> artificial sequence <220> <223> CYP102A1 mutant M850 <400> 24 Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Tyr Gly Glu Leu Lys Asn 1 5 10 15 Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile 20 25 30 Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Leu Val 35 40 45 Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu 50 55 60 Ser Arg Phe Gly Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg Asp 65 70 75 80 Ile Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu Lys Asn Trp 85 90 95 Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met 100 105 110 Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln 115 120 125 Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Gly Asp 130 135 140 Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr 145 150 155 160 Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser 165 170 175 Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln Arg Ala Asn 180 185 190 Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp 195 200 205 Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg Lys 210 215 220 Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly 225 230 235 240 Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr 245 250 255 Gln Ile Ile Thr Phe Leu Ile Ala Gly His Val Thr Thr Ser Gly Leu 260 265 270 Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln 275 280 285 Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser 290 295 300 Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu 305 310 315 320 Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys 325 330 335 Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu 340 345 350 Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly 355 360 365 Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala 370 375 380 Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys 385 390 395 400 Ile Gly Gln Gln Phe Ala Leu Arg Glu Ala Thr Leu Val Leu Gly Met 405 410 415 Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp 420 425 430 Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys Ala 435 440 445 Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu 450 455 460 Gln Ser Ala Lys Lys Val Arg Lys Lys Val Glu Asn Ala His Asn Thr 465 470 475 480 Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr 485 490 495 Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln 500 505 510 Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala 515 520 525 Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala 530 535 540 Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Asp Val Lys 545 550 555 560 Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr 565 570 575 Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys 580 585 590 Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp 595 600 605 Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val 610 615 620 Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser 625 630 635 640 Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala 645 650 655 Lys Met His Gly Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu 660 665 670 Gln Gln Leu Gly Ser Glu Arg Ser Thr Arg His Leu Glu Ile Ala Leu 675 680 685 Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro 690 695 700 Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly Leu 705 710 715 720 Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala 725 730 735 His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr 740 745 750 Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala 755 760 765 Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu 770 775 780 Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met 785 790 795 800 Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu 805 810 815 Phe Ile Ala Leu Leu Pro Ser Ile Ser Pro Arg Tyr Tyr Ser Ile Ser 820 825 830 Ser Ser Pro His Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val 835 840 845 Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala 850 855 860 Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe 865 870 875 880 Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Asp Ser Glu Thr 885 890 895 Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly 900 905 910 Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly 915 920 925 Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu 930 935 940 Tyr Gln Glu Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu 945 950 955 960 His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln 965 970 975 His Val Met Glu Arg Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln 980 985 990 Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Asp 995 1000 1005 Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val Tyr Glu Val Ser 1010 1015 1020 Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg 1025 1030 1035 1040 Tyr Ala Lys Asp Val Trp Ala Gly 1045

Claims (5)

CYP102A1의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 3-히드록시 플로리진(3-hydroxyphlorizin) 제조용 조성물로서,
상기 CYP102A1는 서열번호 1의 아미노산 서열을 포함하고,
상기 CYP102A1의 돌연변이체는 상기 서열번호 1로부터 치환되는 아미노산이
R47L/F81I/F87V/E143G/L188Q/N213S/E267V,
F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R,
D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V,
F42L/R47L/F81I/F87V/E143G/L188Q/E267V,
T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V,
R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R 및
F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R으로 구성되는 군으로부터 선택되는 하나 이상을 포함하는 3-히드록시 플로리진 제조용 조성물.
A composition for preparing 3-hydroxyphlorizin comprising at least one enzyme selected from the group consisting of mutants of CYP102A1,
The CYP102A1 includes the amino acid sequence of SEQ ID NO: 1,
The mutant of CYP102A1 has an amino acid substituted from SEQ ID NO: 1
R47L/F81I/F87V/E143G/L188Q/N213S/E267V;
F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R;
D23G/R47L/F81I/F87V/F107L/D136G/E143G/L188Q/E267V;
F42L/R47L/F81I/F87V/E143G/L188Q/E267V;
T1S/R47L/F81I/F87V/E143G/T146A/L188Q/E267V;
R47L/F81I/F87V/K113N/E143G/L188Q/E267V/N319D/L347I/S383R and
F11Y / R47L / D68G / F81I / F87V / E143G / L188Q / E267V / H408R A composition for preparing 3-hydroxy phlorizin containing at least one selected from the group consisting of.
 제1항에 있어서,
상기 CYP102A1의 돌연변이체는 상기 서열번호 1로부터 치환되는 아미노산이 A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E 인 것을 추가로 포함하는 것을 특징으로 하는 3-히드록시 플로리진 제조용 조성물.
According to claim 1,
In the mutant of CYP102A1, the amino acids substituted from SEQ ID NO: 1 are A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q98 1R/A1008D / H1021Y / Q1022E A composition for preparing 3-hydroxy phlorizin, characterized in that it further comprises.
 제1항 또는 제2항의 3-히드록시 플로리진 제조용 조성물을 포함하는 3-히드록시 플로리진의 생산용 키트.
A kit for producing 3-hydroxy phlorizin comprising the composition for preparing 3-hydroxy phlorizin according to claim 1 or 2.
 제1항 또는 제2항의 3-히드록시 플로리진 제조용 조성물과 플로리진을 반응시키는 단계를 포함하는 3-히드록시 플로리진의 제조방법.
A method for preparing 3-hydroxy phlorizin comprising reacting the composition for preparing 3-hydroxy phlorizin according to claim 1 or 2 with phlorizin.
 제4항에 있어서, NADPH-생성 시스템을 추가하는 단계를 더 포함하는 것을 특징으로 3-히드록시 플로리진의 제조방법.The method of claim 4, further comprising the step of adding a NADPH-generating system.
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KR101446051B1 (en) 2012-07-18 2014-10-01 전남대학교산학협력단 Novel preparation method of metabolites of genistein using bacterial cytochrome P450 and composition therefor
KR101992247B1 (en) 2017-09-27 2019-06-24 전남대학교산학협력단 Composition for 3-hydroxyl phloretin production

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KR101446051B1 (en) 2012-07-18 2014-10-01 전남대학교산학협력단 Novel preparation method of metabolites of genistein using bacterial cytochrome P450 and composition therefor
KR101992247B1 (en) 2017-09-27 2019-06-24 전남대학교산학협력단 Composition for 3-hydroxyl phloretin production

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