KR102259266B1 - Composition for preparing neoeriocitrin dihydrochalcone - Google Patents

Composition for preparing neoeriocitrin dihydrochalcone Download PDF

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KR102259266B1
KR102259266B1 KR1020190070084A KR20190070084A KR102259266B1 KR 102259266 B1 KR102259266 B1 KR 102259266B1 KR 1020190070084 A KR1020190070084 A KR 1020190070084A KR 20190070084 A KR20190070084 A KR 20190070084A KR 102259266 B1 KR102259266 B1 KR 102259266B1
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윤철호
응우옌티흥하
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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/34—Sugar alcohols
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)

Abstract

본 발명의 일 실시예는 CYP102A1의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 네오에리오시트린 디하이드로칼콘(neoeriocitrin dihydrochalcone) 제조용 조성물, 키트, 및 이를 이용하는 네오에리오시트린 디하이드로칼콘의 제조방법을 제공한다. One embodiment of the present invention provides a composition, kit, and neoeriocitrin dihydrochalcone for preparing neoeriocitrin dihydrochalcone containing one or more enzymes selected from the group consisting of mutants of CYP102A1, and neoeriocitrin dihydrochalcone using the same. A manufacturing method is provided.

Description

네오에리오시트린 디하이드로칼콘 제조용 조성물{Composition for preparing neoeriocitrin dihydrochalcone}Composition for preparing neoeriocitrin dihydrochalcone {Composition for preparing neoeriocitrin dihydrochalcone}

본 발명은 박테리아 시토크롬 P450을 이용하여 나린진 디하이드로칼콘(naringin dihydrochalcone)으로부터 그의 대사체인 네오에리오시트린 디하이드로칼콘(neoeriocitrin dihydrochalcone)을 제조하기 위한 신규한 조성물에 관한 것이다.The present invention relates to a novel composition for preparing its metabolite neoeriocitrin dihydrochalcone from naringin dihydrochalcone using bacterial cytochrome P450.

나린진 디하이드로칼콘(naringin dihydrochalcone; 나린진 DC)은 감귤류에서 발견되는 쓴맛이 나는 화합물인 나린진에서 생산한 인공 감미료이다. 나린진 DC는 감귤류 주스에서 쓴맛을 최소화하는 방법을 찾기 위해 미국 농무성 연구 프로그램의 일환으로 1960년대에 네오헤스페리진 디하이드로칼콘(neohesperidin dihydrochalcone)과 동시에 발견된 플로레틴 배당체(phloretin glycoside)의 한 종류이다. 나린진을 수산화칼륨 또는 다른 강염기로 처리한 다음 촉매적으로 수소화하면 디하이드로칼콘이되어 임계 농도에서 설탕보다 약 500-700 배 더 단맛을 낸다. Naringin dihydrochalcone (naringin DC) is an artificial sweetener produced from naringin, a bitter-tasting compound found in citrus fruits. Naringin DC is a class of phloretin glycoside discovered concurrently with neohesperidin dihydrochalcone in the 1960s as part of a US Department of Agriculture research program to find a way to minimize bitterness in citrus juice. . When naringin is treated with potassium hydroxide or another strong base and then catalytically hydrogenated, it becomes a dihydrochalcone, which at critical concentrations is about 500-700 times sweeter than sugar.

나린진 DC는 네오헤스페리딘 디하이드로칼콘과 비슷한 형태의 감미료로서, 청결한 맛, 저칼로리, 무해함 및 안전성과 같은 많은 바람직한 특성을 지니고 있어 식품 및 의약품 모두에 사용할 수 있다. 나린진 DC는 강렬한 단맛은 감미료로 사용되는 것 외에도 식품 및 의약품의 쓴맛을 가리는 데에도 유용하다. 또한, 나린진 DC는 해당 플라바논 나린진보다 더 큰 자유 라디칼 소거능을 나타내는 강력한 항산화제이다. 따라서, 나린진 DC는 비칼로리 감미료와 기능성 식품 및 음료, 건강 기능 식품 및 의약품에 응용 가능성이 있는 항산화제로 작용할 수 있는 역량을 갖추고 있다. 또한, 나린진 DC는 베타 아밀로이드(Aβ) 병리, 신경 염증 및 신경 발생을 포함한 여러 표적에 대한 알츠하이머 병 치료의 잠재적 치료제로 사용될 가능성이 제안되었다. Naringin DC is a sweetener similar to neohesperidin dihydrochalcone, and has many desirable properties such as clean taste, low calorie, harmlessness and safety, so it can be used in both food and pharmaceutical products. Naringin DC's intense sweetness is not only used as a sweetener, but is also useful in masking the bitter taste of foods and pharmaceuticals. In addition, naringin DC is a powerful antioxidant that exhibits greater free radical scavenging activity than the corresponding flavanone naringin. Therefore, Naringin DC has the ability to act as a non-calorie sweetener and antioxidant with potential applications in functional foods and beverages, health functional foods and pharmaceuticals. In addition, naringin DCs have been proposed for potential therapeutic use in the treatment of Alzheimer's disease against multiple targets including beta-amyloid (Aβ) pathology, neuroinflammation and neurogenesis.

그러나, 효소전환 방법으로 나린진 DC로부터 네오에리오시트린 디하이드로칼콘(neoeriocitrin dihydrochalcone)을 생산하는 방법은 아직까지 보고된 바 없다. However, a method for producing neoeriocitrin dihydrochalcone from naringin DC by enzymatic conversion has not yet been reported.

시토크롬 P450 효소(P450s 또는 CYPs)는 고세균(archaea)에서부터 박테리아, 곰팡이, 식물, 동물 및 사람에 이르기까지 자연 전반에 걸쳐 매우 다양한 산화반응의 촉매로써 역할을 하는 효소들로 구성되는 대형 패밀리이다(http://drnelson.utmen.edu/CytochromeP450.html). 이들의 촉매작용의 다양성과 광범위한 기질로 인해 P450s은 의약품, 천연물 소재 등을 포함하는 정밀화학물질의 생산에 생물학적 촉매로써의 유용성이 크다. 그러나 이와 같은 다양한 생물공학 분야에서의 포유동물의 시토크롬 P450 효소들의 잠재적인 유용성에도 불구하고, 안정성, 촉매활성 및 입수가능성이 낮아 생물학적 촉매로써 적합하지 않은 면이 있다. Cytochrome P450 enzymes (P450s or CYPs) are a large family of enzymes that act as catalysts for a wide variety of oxidation reactions throughout nature, from archaea to bacteria, fungi, plants, animals and humans. ://drnelson.utmen.edu/CytochromeP450.html). Due to their diversity of catalysis and a wide range of substrates, P450s have great utility as biological catalysts in the production of fine chemicals including pharmaceuticals and natural materials. However, despite the potential usefulness of mammalian cytochrome P450 enzymes in various fields of biotechnology, they are not suitable as biological catalysts due to their low stability, catalytic activity and availability.

음식물로 섭취하는 식물에 존재하는 식물화학물질(phytochemicals) 또는 약물이나 효능, 독성, 약물동력학 등에 대한 연구를 하기 위해서는 대량의 순수한 대사산물이 필요하게 된다. 또한 대사산물 자체가 생리활성을 가질 경우, 그 대사산물을 직접 생체 내에 투여하는 것이 큰 이점을 가져 올 수 있으며, 따라서 이를 대량으로 제조하는 것이 중요하다. In order to study phytochemicals or drugs, efficacy, toxicity, pharmacokinetics, etc. present in plants consumed as food, a large amount of pure metabolites are required. In addition, when the metabolite itself has physiological activity, direct administration of the metabolite in vivo can bring a great advantage, and therefore it is important to mass-produce it.

그러나, 순수한 대사산물을 화학적으로 합성하는 것에 여러 가지 문제점이 있으므로, 대사산물의 화학합성의 대안으로 바이오 기능성소재 및 약물이나 약물 후보의 대사산물을 만들기 위하여 P450s을 이용한다. 대장균이나 곤충세포에서 발현된 사람 P450s를 이용하여 대사산물을 제조하는 것이 보고된 바 있다. 그러나, 이러한 시스템들은 한정된 안정성 및 느린 반응속도 등으로 인하여 비용이 많이 들고 생산성이 낮다. 사람에서의 대사산물을 제조하기 위한 대안으로 원하는 촉매활성을 갖는 조작된 박테리아 P450 효소들을 사용하는 방법이 제안되었다. However, since there are several problems in chemically synthesizing pure metabolites, P450s are used to make biofunctional materials and metabolites of drugs or drug candidates as an alternative to chemical synthesis of metabolites. It has been reported that metabolites are produced using human P450s expressed in E. coli or insect cells. However, these systems are expensive and low in productivity due to limited stability and slow reaction rates. As an alternative to the production of metabolites in humans, a method using engineered bacterial P450 enzymes with desired catalytic activity has been proposed.

한편, 바실러스 메가테리움(Bacillus megaterium)으로부터 유래한 P450 BM3 (CYP102A1)의 헴 영역(heme domain)은 모노옥시게나제 활성(monooxygenase activity)을 갖는데, CYP4A(지방산 히드록시라제) 패밀리의 포유동물 멤버와 아주 유사하다. 자연적으로, 포유동물-유사 디플라빈 환원효소(mammalian-like diflavin reductase) 기능을 갖는 CYP102A1 환원효소 도메인을 P450 헴 영역(heme domain) 도메인의 C-말단에 융합된 단일 폴리펩티드로 만들어져있다. 이러한 두 효소 활성의 융합은 가용성 CYP102A1을 이상적인 포유동물 모델, 특히 사람 P450 효소의 이상적인 모델로 만든다. 합리적 디자인(rational design) 또는 유도진화(directed evolution)를 통하여 유전공학적으로 제조되는 CYP102A1 돌연변이체들은 몇몇 사람 P450의 기질을 산화하여 더 활성이 높은 대사산물로 만든다는 것이 보고되었다. 이러한 최근의 진보로 CYP102A1 돌연변이체들이 약물의 발견과 합성을 위한 생물 촉매로서 개발될 수 있다는 것이 제안되었다. 아주 최근에는 몇몇 선택된 돌연변이는 CYP102A1 효소가 약물의 사람에서의 대사산물을 생산하도록 한다는 것이 보고되었다. 더욱이, CYP102A1은 다양한 기질에 작용할 수 있는 다목적 모노옥시게나제이며 생물 공학적 이용에 적절하다. On the other hand, the heme domain of P450 BM3 (CYP102A1) derived from Bacillus megaterium has monooxygenase activity, a mammalian member of the CYP4A (fatty acid hydroxylase) family. very similar to Naturally, the CYP102A1 reductase domain with mammalian-like diflavin reductase function is made into a single polypeptide fused to the C-terminus of the P450 heme domain domain. The fusion of these two enzymatic activities makes soluble CYP102A1 an ideal mammalian model, particularly the human P450 enzyme. It has been reported that CYP102A1 mutants, which are genetically engineered through rational design or directed evolution, oxidize some human P450 substrates to form more active metabolites. These recent advances have suggested that CYP102A1 mutants can be developed as biocatalysts for drug discovery and synthesis. Very recently, it has been reported that several selected mutations cause the CYP102A1 enzyme to produce a human metabolite of the drug. Moreover, CYP102A1 is a versatile monooxygenase that can act on a variety of substrates and is suitable for bioengineering applications.

본 발명의 명세서에 인용되는 선행문헌의 내용은 모두 본 명세서에 도입된다. 또한 이곳에 기재된 정보들은 단지 본 발명의 기술적 배경에 대한 이해를 돕고자 하는 것으로서, 본 발명에 대한 선행기술로써 작용할 수 없음은 당연하다. All contents of the prior literature cited in the specification of the present invention are incorporated herein. In addition, the information described herein is only intended to help the understanding of the technical background of the present invention, and it is natural that it cannot act as a prior art for the present invention.

Whitehouse CJ, Bell SG, Wong LL. P450(BM3) (CYP102A1): connecting the dots. Chem Soc Rev. 41(3):1218-1260, 2012 Whitehouse CJ, Bell SG, Wong LL. P450(BM3) (CYP102A1): connecting the dots. Chem Soc Rev. 41(3):1218-1260, 2012 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(7):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(7):1313-1322, 2014 Yun CH, Kim KH, Kim DH, Jung HC, Pan JG. The bacterial P450 BM3: a prototype for a biocatalyst with human P450 activities. 25(7):289-298, 2007 Yun CH, Kim KH, Kim DH, Jung HC, Pan JG. The bacterial P450 BM3: a prototype for a biocatalyst with human P450 activities. 25(7):289-298, 2007 Whitehouse CJ, Bell SG, Wong LL. P450(BM3) (CYP102A1): connecting the dots. Chem Soc Rev. 41(3):1218-1260, 2012 Whitehouse CJ, Bell SG, Wong LL. P450(BM3) (CYP102A1): connecting the dots. Chem Soc Rev. 41(3):1218-1260, 2012

본 발명의 일 과제는 나린진 DC의 대사산물인 네오에리오시트린 디하이드로칼콘을 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 producing a large amount of neoeriocitrin dihydrochalcone, a metabolite of naringin DC, and a mutant thereof.

본 발명의 발명자들은 상기 과제를 해결하고자 연구한 결과, 박테리아 P450 효소인 CYP102A1 돌연변이체가 나린진 DC의 산화를 매개하여 특정 대사산물, 특히, 네오에리오시트린 DC을 생산하는데 이용될 수 있다는 것을 발견하고, 이를 기초로 본 발명을 완성하였다. As a result of research to solve the above problems, the inventors of the present invention found that CYP102A1 mutant, a bacterial P450 enzyme, can be used to mediate oxidation of naringin DC to produce specific metabolites, in particular, neoeriocitrin DC, Based on this, the present invention was completed.

본 발명의 일 양태는 CYP102A1의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 네오에리오시트린 디하이드로칼콘(neoeriocitrin dihydrochalcone) 제조용 조성물로서, 상기 CYP102A1는 서열번호 1의 아미노산 서열을 포함하고, 상기 CYP102A1의 돌연변이체는 상기 서열번호 1로부터 치환되는 아미노산이 R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V/E143G/L188Q/N213S/E267V 및 F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R으로 구성되는 군으로부터 선택되는 하나 이상을 포함하는 네오에리오시트린 디하이드로칼콘 제조용 조성물을 제공한다. One aspect of the present invention is a composition for preparing neoeriocitrin dihydrochalcone comprising one or more enzymes selected from the group consisting of mutants of CYP102A1, wherein CYP102A1 comprises the amino acid sequence of SEQ ID NO: 1, The mutant of CYP102A1 has amino acids substituted from SEQ ID NO: 1 R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V /E143G/L188Q/N213S/E267V and F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R provides a composition for preparing neo-eriocitrin dihydrochalcone comprising at least one selected from the group consisting of.

본 발명의 일 실시예에 따르면, 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/Q1022E인 것을 추가로 포함할 수 있다. According to an embodiment of the present invention, in the mutant of CYP102A1, the amino acid substituted from SEQ ID NO: 1 is A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/ P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E.

본 발명의 다른 일 양태는 상기 네오에리오시트린 디하이드로칼콘 제조용 조성물을 포함하는 네오에리오시트린 디하이드로칼콘의 생산용 키트를 제공한다. Another aspect of the present invention provides a kit for the production of neo eriocitrin dihydrochalcone comprising the composition for preparing the neoeriocitrin dihydrochalcone.

본 발명의 또 다른 일 양태는 상기 네오에리오시트린 디하이드로칼콘 제조용 조성물과 나린진 디하이드로칼콘을 반응시키는 단계를 포함하는 네오에리오시트린 디하이드로칼콘의 제조방법을 제공한다. Another aspect of the present invention provides a method for preparing neo eriocitrin dihydrochalcone, comprising reacting the composition for preparing neoeriocitrin dihydrochalcone with naringin dihydrochalcone.

본 발명의 일 실시예에 따르면, 상기 제조방법은 NADPH-생성 시스템을 추가하는 단계를 더 포함할 수 있다. According to an embodiment of the present invention, the manufacturing method may further include adding a NADPH-generating system.

본 발명의 일 실시예의 박테리아 시토크롬 P450, 즉, CY102A1의 돌연변이체를 이용하여 나린진 DC의 대사산물인 네오에리오시트린 DC를 특이적으로 대량 생산할 수 있다. 상기 네오에리오시트린 DC는 화학적 합성방법이 보고된 바 없어, 본 발명의 일 실시예는 이의 대량 생산을 위한 대안을 제공할 수 있다. Neoeriocitrin DC, a metabolite of naringin DC, can be specifically mass-produced by using the bacterial cytochrome P450, that is, a mutant of CY102A1 of an embodiment of the present invention. Since the neoeriocitrin DC has not been reported as a chemical synthesis method, an embodiment of the present invention may provide an alternative for mass production thereof.

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 configuration of the invention described in the detailed description or claims of the present invention.

도 1은 본 발명의 일 실시예에 따른 CYP102A1 야생형 및 이의 돌연변이체에 의한 나린진 DC의 대사산물 생성속도를 나타낸다.
도 2는 본 발명의 일 실시예에 따른 CYP102A1 야생형 및 이의 돌연변이체에 의하여 생성된 나린진 DC 대사산물의 HPLC 크로마토그램(285 nm에서의 UV 흡광도를 측정) 결과를 나타낸다(피크: CYP102A1 돌연변이체에 의해 생성된 대사산물 피크와 체류 시간을 비교하여 확인한 것, 화살표: 기질인 나린진 DC 및 주요 산물인 네오에리오시트린 DC를 표시함).
도 3은 나린진 DC 및 본 발명의 일 실시예에 따른 CYP102A1 돌연변이체에 의하여 생성된 그의 대사산물의 LC-MS 용출 프로파일을 나타낸다.
도 4는 본 발명의 일 실시예에 따른 CYP102A1 돌연변이체에 의하여 생성된 대사산물의 양성자 NMR 분석 결과(a) 및 이에 따른 구조분석 결과(b)와, COSY-2D NMR 분석 결과(c) 및 이에 따른 구조분석 결과(d)를 나타낸다.
도 5는 본 발명의 일 실시예에 따른 CYP102A1 돌연변이체 M16V3, G1, M179, M221에 의한 네오에리오시트린 DC 산물의 생성효율을 비교한 속도론적 매개변수 분석결과를 나타낸다.
도 6은 야생형 CYP102A1의 아미노산 서열을 나타낸다.
도 7은 야생형 CYP102A1의 돌연변이체인 M16의 아미노산 서열을 나타낸다.
도 8은 야생형 CYP102A1의 돌연변이체인 M16V3의 아미노산 서열을 나타낸다.
도 9 내지 도 11은 야생형 CYP102A1의 돌연변이체인 G1, M179 및 M221의 아미노산 서열을 나타낸다. 1 shows the metabolite production rate of naringin DC by CYP102A1 wild-type and mutants thereof according to an embodiment of the present invention.
2 shows the HPLC chromatogram (measurement of UV absorbance at 285 nm) results of naringin DC metabolites produced by CYP102A1 wild-type and mutants thereof according to an embodiment of the present invention (peak: by CYP102A1 mutants) Comparison of the resulting metabolite peak and retention time, arrows: indicate substrate naringin DC and major product neoeryocitrin DC).
3 shows the LC-MS elution profile of naringin DC and its metabolites produced by the CYP102A1 mutant according to an embodiment of the present invention.
4 is a proton NMR analysis result (a) and structural analysis result (b) of the metabolite produced by the CYP102A1 mutant according to an embodiment of the present invention (b), COSY-2D NMR analysis result (c) and this Structural analysis result (d) is shown.
5 shows the results of analysis of kinetic parameters comparing the generation efficiency of neoeriocitrin DC products by CYP102A1 mutants M16V3, G1, M179, and M221 according to an embodiment of the present invention.
6 shows the amino acid sequence of wild-type CYP102A1.
7 shows the amino acid sequence of M16, a mutant of wild-type CYP102A1.
8 shows the amino acid sequence of M16V3, a mutant of wild-type CYP102A1.
9 to 11 show the amino acid sequences of G1, M179 and M221, which are mutants 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. Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and therefore is not limited to the embodiments described herein.

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, bonded)" with another part, it is not only "directly connected", but also "indirectly connected" with another member in the middle. "Including the case. In addition, when a part "includes" a certain component, this means that other components may be further provided, not excluding other components, unless specifically stated to the contrary.

본 명세서에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. The terms used in the present specification are only used to describe specific embodiments, and are not intended to limit the present invention.

본 발명의 일 양태는 박테리아 시토크롬 P450의 돌연변이체로 구성되는 군으로부터 선택되는 하나 이상의 효소를 포함하는 네오에리오시트린 디하이드로칼콘 제조용 조성물을 제공한다. One aspect of the present invention provides a composition for preparing neoeriocitrin dihydrochalcone comprising one or more enzymes selected from the group consisting of mutants of bacterial cytochrome P450.

상기 네오에리오시트린 디하이드로칼콘 제조용 조성물에 포함된 박테리아 시토크롬 P450 효소 및 이의 돌연변이체는, 하기 화학식 1과 같이, 나린진 디하이드로칼콘(나린진 DC)를 기질로 하여 대사물질인 네오에리오시트린 디하이드로칼콘(네오에리오시트린 DC)를 선택적이며 안정적이고 고효율로 대량생산할 수 있다. The bacterial cytochrome P450 enzyme and its mutants included in the composition for preparing the neo-eryocitrin dihydrochalcone, as shown in the following Chemical Formula 1, use naringin dihydrochalcone (naringin DC) as a substrate and neo-eryocitrin dihydro, a metabolite It is possible to selectively, stably, and efficiently mass-produce chalcone (Neoeriocitrin DC).

[화학식 1][Formula 1]

Figure 112019060533281-pat00001
Figure 112019060533281-pat00001

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 CYP102A1, which is a bacterial cytochrome P450 BM3. 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 a 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 nonpolar amino acids, and they have similar properties to each other. Non-charged 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 an individual in which a point mutation has occurred based on the amino acid sequence of wild-type CYP102A or a fusion chimera 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, etc. However, the present invention 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, for example, 70% or more, for example, 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 chimeric CYP102A1 may include a CYP102A1 chimera and a point mutant of the 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.

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 existing in nature other than the previously known CYP102A1 as a result of confirming the nucleotide sequence of the CYP102A1 gene of Bacillus sp. collected and deposited in nature. In the chimeric CYP102A1, the point mutants of wild-type CYP102A1 were expressed in large quantities in E. coli, and one with a large catalytic activity was selected among the mutants, and the heme region of the selected CYP102A1 mutant was fused with the reductase region of the natural variant of wild-type CYP102A1 can be manufactured. In addition, the prepared point mutants of chimeric CYP102A1 can be expressed in large amounts in E. coli to select those having high catalytic activity to select mutants of chimeric CYP102A1.

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 mutant of CYP102A1 may be represented in the order of '(amino acid before mutation), (position of amino acid), (amino acid substituted due to mutation)'. For example, R47L refers to a CYP102A1 mutant in which arginine (R), which is the 47th amino acid of SEQ ID NO: 1, which is the amino acid sequence of wild-type CYP102A1, is substituted with leucine (L) either naturally or artificially by mutation induction. In the case of one or more amino acids substituted by mutation in the mutant, it may be represented by '/'. For example, in R47L/F81I/F87V, arginine (R), the 47th amino acid of SEQ ID NO: 1, is substituted with leucine (L), and phenylalanine (F), the 81st amino acid, of SEQ ID NO: 1 is substituted with isoleucine (I) and means 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 in the present invention to indicate chimeric CYP102A1 or mutant mutations of chimeric CYP102A1.

상기 야생형 CYP102A1의 돌연변이체는 야생형 CYP102A1의 점 돌연변이체일 수 있다. 예를 들어, 야생형 CYP102A1의 점 돌연변이체인 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V/E143G/L188Q/N213S/E267V 및/또는 F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R의 야생형 CYP102A1의 돌연변이체일 수 있다. The mutant of wild-type CYP102A1 may be a point mutant of wild-type CYP102A1. For example, R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V of wild-type CYP102A1 that are point mutants of wild-type CYP102A1 /L188Q/N213S/E267V and/or F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R 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/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V/E143G/L188Q/N213S/E267V 및/또는 F11Y/R47L/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 mutant of wild-type CYP102A1 may include chimeric CYP102A1 in which the ham region of the point mutant of wild-type CYP102A1 and the reducing region of the natural variant of wild-type CYP102A1 are fused. For example, R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V of wild-type CYP102A1 that are point mutants of wild-type CYP102A1 The heme region of /L188Q/N213S/E267V and/or F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R and the reducing region of the native variant of wild-type CYP102A1 A474V/E558D/T664A/P675L/A678E/E687A/A741G /K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E may be a 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 mutant of wild-type CYP102A1 above, the mutant of wild-type CYP102A1 is a point mutant of wild-type CYP102A1, a natural mutant of wild-type CYP102A1, the heme region of a point mutant of wild-type CYP102A1, and reductase of a natural mutant of wild-type CYP102A1. chimeric CYP102A1 and point mutants of chimeric CYP102A1 fused regions.

예를 들어, 야생형 CYP102A1의 돌연변이체는 서열번호 1로 표시되는 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/1021Y/Q1022E, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/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/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/Q1022E로 구성된 군으로부터 선택되는 하나 이상일 수 있다. For example, a mutant of wild-type CYP102A1 is R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H of wild-type CYP102A1 represented by SEQ ID NO: 1 /E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/1021Y/Q1022E, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/A474V/E558D/T664A/P675L/A664A /A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/E143G/L188Q/N64213S/E267E58V/ /A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F11Y/R47L/F81I/L188V/E143G/F87V/E143G/F87V/E143G /E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E.

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 method for synthesizing a peptide 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 prepared by a method such as cleavage 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 prepared as a natural protein, and can be prepared by a recombinant method of culturing and recovering cells transformed with DNA encoding wild-type CYP102A1, a point mutant of wild-type CYP102A1, chimeric CYP102A1 and a point mutant of chimeric CYP102A1. may be Specifically, the enzyme of the present invention is inserted by inserting a nucleic acid molecule encoding the enzyme of the present invention into an appropriate expression vector, culturing the transformant prepared 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, a cosmid, a viral particle or a 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, and pH can be appropriately selected without excessive experimentation in the field to which the present invention pertains. 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 comprise a promoter operably linked to a nucleic acid sequence encoding wild-type CYP102A1, a point mutant of wild-type CYP102A1, chimeric CYP102A1 and a point mutant of chimeric CYP102A1, which direct mRNA synthesis. Various promoters recognized by host cells are known. Promoters suitable for use in prokaryotic hosts include the β-lactamase and lactose promoter systems, the alkaline fostapase, the tryptophan promoter system, and 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 네오에리오시트린 디하이드로칼콘 제조용 조성물과 나린진 DC를 반응시키는 단계를 포함하는 네오에리오시트린 디하이드로칼콘의 제조방법을 제공한다. Another aspect of the present invention comprises the step of reacting naringin DC with a composition for preparing neoeriocitrin dihydrochalcone comprising one or more enzymes selected from the group consisting of CYP102A1 mutants according to an embodiment of the present invention Provided is a method for preparing neoeryocitrin dihydrochalcone.

본 발명의 일 실시예에 따르면, 상기 야생형 CYP102A1의 돌연변이체는 나린진 DC를 기질로 하여 나린진 DC를 보다 효율적으로 생산할 수 있다. According to an embodiment of the present invention, the wild-type CYP102A1 mutant can more efficiently produce naringin DC using naringin DC as a substrate.

상기 야생형 CYP102A1의 돌연변이체와 나린진 DC의 반응 시간은, 예를 들어, 1 분 내지 6 시간, 예를 들어, 1 시간 내지 4 시간, 예를 들어, 3 시간일 수 있으나, 이에 한정되는 것은 아니다. 상기 야생형 CYP102A1 및 이의 돌연변이체를 반응 기질인 나린진 DC와 반응 시 나린진 DC의 농도 및 효소의 농도는 적절하게 변경할 수 있다. The reaction time between the wild-type CYP102A1 mutant and naringin DC may be, for example, 1 minute to 6 hours, for example, 1 hour to 4 hours, for example, 3 hours, but is not limited thereto. When the wild-type CYP102A1 and its mutant are reacted with naringin DC as a reaction substrate, the concentration of naringin DC and the concentration of the enzyme can 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 1 μM 내지 1 mM, 예를 들어, 100 μM로 포함될 수 있다. The preparation 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 to which the present invention pertains, for example, glucose 6-phosphate, NADP- (nicotinamide adenine dinucleotide phosphate) and yeast glucose 6-phosphate dehydrogena. It may be used (glucose 6-phosphate dehydrogenase), but is not limited thereto. The NADPH-generating system may be comprised between 1 μM and 1 mM, for example 100 μM.

본 발명의 다른 일 양태는 상기 네오에리오시트린 DC 제조용 조성물을 포함하는 네오에리오시트린 DC 생산용 키트를 제공한다. Another aspect of the present invention provides a kit for producing DC Neo Eriocitrin comprising the composition for producing DC Neo Eriocitrin.

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

상기 야생형 CYP102A1의 돌연변이체는, 예를 들어, 서열번호 1로 표시되는 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V/E143G/L188Q/N213S/E267V 및/또는 F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R일 수 있다. The mutant of wild-type CYP102A1 is, for example, R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A of wild-type CYP102A1 represented by SEQ ID NO: 1 , R47L/F81I/F87V/E143G/L188Q/N213S/E267V and/or F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R.

상기 야생형 CYP102A1의 돌연변이체는, 예를 들어, 서열번호 1로 표시되는 야생형 CYP102A1의 R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/1021Y/Q1022E, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/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/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/Q1022E로 구성된 군으로부터 선택되는 하나 이상일 수 있다. The mutant of wild-type CYP102A1 is, for example, R47L/F81I/F87V/E143G/L188Q/E267V/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R8 of wild-type CYP102A1 represented by SEQ ID NO: 1 /R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/1021Y/Q1022E, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/A78474V/E558D/ /E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, R47L/F81I/F87V/E143G/L188267V/N64213S/L188267V/N64213S /P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E, F11Y/E47L/F81E143G/F87V /A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E may be one or more selected from the group consisting of: .

상기 네오에리오시트린 DC 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 neoeriocitrin DC production kit may further include a NADPH-generating system. The NADPH-producing system is a system known in the art to which the present invention pertains, for example, glucose 6-phosphate, NADP- (nicotinamide adenine dinucleotide phosphate) and yeast glucose 6-phosphate dehydrogena. It may be used (glucose 6-phosphate dehydrogenase), but is not limited thereto.

상기 네오에리오시트린 DC 생산용 키트는 그 외에 반응을 진행시키는데 필요한 시약을 추가로 포함할 수 있다. The neoeriocitrin DC production kit may further include reagents necessary to proceed with the reaction.

본 발명의 일 실시예에 따른 네오에리오시트린 DC 제조용 조성물, 키트, 및 이의 제조방법은 나린진 DC의 대사산물인 네오에리오시트린 DC를 경제적이면서 고효율로 대량생산할 수 있어, 기능성 바이오소재 및 약물의 개발 과정에서 네오에리오시트린 DC의 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 preparing neoeriocitrin DC according to an embodiment of the present invention can mass-produce neoeriocitrin DC, which is a metabolite of naringin DC, in an economical and high-efficiency manner, so that During the development process, it can be used to evaluate the efficacy, toxicity, pharmacokinetics, etc. of neoeriocitrin DC, 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 way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

실시예Example

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 Methods 의 construction of BM3 Mutants by Site-directed Mutagenesis 참조), M16(서열번호 2, 도 7)으로 명명하였다(하기 표 2 참조). The site-directed mutant M16 of wild-type CYP102A1 was prepared by the same method as that prepared by 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 Drug Metabolism and Disposition 36(11):2166-2170. See page 2 of the paper (page 2167) Construction of BM3 Mutants by Site-directed Mutagenesis), M16 (SEQ ID NO: 2, Fig. 7) (see Table 2 below).

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, the primers listed in Table 1 below and PCR primers for mutagenesis (XENOTECH, Korea) were used. Codons for amino acid substitution 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. FW 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, it was confirmed by requesting DNA sequencing (Genotech, Korea) whether the desired mutation 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 by 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 서열 order 서열번호Sequence number BamH I forwardBamH I forward 5'- AGC GGA TCC ATG ACA ATT AAA GAA ATG CCT C -3'5'- AGC GGA TC C ATG ACA ATT AAA GAA ATG CCT C -3' 33 Sac I reverseSac I reverse 5'- ATC GAG CTC GTA GTT TGT AT -3'5'- ATC GAG CTC GTA GTT TGT AT -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

야생형 CYP102A1의
점 돌연변이체of wild-type CYP102A1
point mutant 야생형 CYP102A1(서열번호 1)의
아미노산 치환 부위of wild-type CYP102A1 (SEQ ID NO: 1)
amino acid substitution site 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 V3)의 환원효소 영역을 융합하여 선택적인 키메라 CYP102A1 단백질인 M16V3을 구축하였다. 야생형 CYP102A1의 점 돌연변이체는 상기 실시예 1의 M16을 사용하였고, 자연적 변형체는 Bacillus megaterium 종의 자연적 변이체(하기 표 3 참조)을 사용하였다. M16V3, a selective chimeric CYP102A1 protein, was constructed by fusing the heme region of the point mutant of wild-type CYP102A1 with the reductase region of the natural variant V3 of wild-type CYP102A1. M16 of Example 1 was used as the point mutant of wild-type CYP102A1, and a natural variant of Bacillus megaterium species (see Table 3 below) was used as the natural variant.

헴 영역과 환원효소 영역을 위하여 BamHI/SacI 및 SacI/XhoI을 사용하여 만들어진 키메라 CYP102A1 M16V3을 발현 벡터 pCW 벡터로 클로닝하였다. 이를 다시 돌연변이 시켜 키메라 CYP102A1 M16V3의 3종의 점 돌연변이체 G1, M179 및 M221을 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). Chimeric CYP102A1 M16V3 made using BamHI/SacI and SacI/XhoI for the heme region and reductase region was cloned into the expression vector pCW vector. This was mutated again to prepare three point mutants G1, M179 and M221 of the chimeric CYP102A1 M16V3 (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).

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 야생형 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 point mutants thereof into the expression vector pCWori (Dr. FW Dahlquist, University of California, Santa Barbara, CA) or pSE420 (Invitrogen) was amplified from pCWBM3. Oligonucleotide assembly was performed using the primers shown 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 this plasmid, which was also used to express a mutant protein of wild-type CYP102A1. After mutagenesis, it was confirmed by requesting DNA sequencing (Genotech, Korea) whether the desired mutation occurred.

균주strain 변종명Variant name 등록번호(Accession Number)Accession 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(100 μg/ml)이 추가된 5 ml Luria-Bertani 배지에 접종하여 37 ℃에서 배양하고, 이 배양물을 암피실린(100 μg/ml)이 추가된 250 ml Terrific Broth 배지에 접종하여 37 ℃에서 250 rpm으로 흔들어 주며 OD600에서 0.8 정도가 될 때까지 배양하고, isopropyl-β를 최종 농도 0.5 mM이 되도록 첨가하여 유전자 발현을 유도하였다. δacid(0.1 mM)을 첨가하였다. 발현이 유도된 후, 30 ℃에서 추가로 36 시간 더 배양하고 원심분리(15 분, 5000 g, 4 ℃)하여 세포를 수확하였다. 세포 펠렛을 TES 완충액(100 mM Tris-HCL, pH7.6, 500 mM sucrose, 0.5 mM EDTA)에 재현탁시키고 초음파분해로 세포를 용해시켰다(sonicator; Misonix, Inc., Farmingdale, NY). 세포용해물을 100,000 g, 90 분, 4 ℃에서 원심분리한 후, 가용성 시토졸 분액(soluble cytosolic fraction)을 모아 활성을 측정하였다. 시토졸 분액을 50 mM 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 a plasmid containing the genes of wild-type CYP102A1 and wild-type CYP102A1 mutants (Kim et al., Protein Expr Purif. 57:188-200., 2008). An appropriate amount from one colony was inoculated into 5 ml Luria-Bertani medium supplemented with ampicillin (100 μg/ml) and cultured at 37° C., and the culture was cultured in 250 ml Terrific Broth medium supplemented with ampicillin (100 μg/ml). was inoculated at 37 °C, shaken at 250 rpm, and incubated at an OD of 600 to about 0.8, and isopropyl-β was added to a final concentration of 0.5 mM to induce gene expression. δacid (0.1 mM) was added. After the expression was induced, the cells were harvested by incubation at 30° C. for an additional 36 hours and centrifugation (15 min, 5000 g, 4° C.). The cell pellet was resuspended in TES buffer (100 mM Tris-HCL, pH7.6, 500 mM sucrose, 0.5 mM EDTA) and lysed by sonication (sonicator; Misonix, Inc., Farmingdale, NY). After centrifuging the cell lysate at 100,000 g, 90 minutes, and 4°C, the soluble cytosolic fraction was collected and activity was measured. The cytosol fraction was dialyzed in 50 mM potassium phosphate buffer (pH 7.4) and stored at -80 °C, and those within 1 month after preparation were used for the experiment.

CYP102A1의 농도는 CO-difference 스펙트럼으로부터 결정하였으며, 이때 사용한 ε은 91 mM/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 300-700 nM P450을 얻을 수 있었다. 야생형 CYP102A1 및 야생형 CYP102A1의 돌연변이체의 발현정도는 1.0~2.0 nmol P450/mg cytosolic protein의 범위였다. 제조된 야생형 CYP102A1의 돌연변이체들 중에서 사람에 있어서의 기질에 대한 촉매 활성이 큰 키메라 CYP102A1의 돌연변이체인 M16V3 및 이의 돌연변이체 G1, M179 및 M221을 선택하였다(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 the ε used 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 mutant typically yielded 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, M16V3, which is a mutant of the chimeric CYP102A1 having high catalytic activity against a human substrate, and its mutants G1, M179 and M221 were selected (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).

하기 표 4에 야생형 CYP102A1의 돌연변이체에서의 아미노산이 치환된 부위를 나타내었다:Table 4 below shows the amino acid substitution sites in the mutant of wild-type CYP102A1:

키메라 CYP102A1 및 이의 점 돌연변이체Chimeric CYP102A1 and point mutants thereof 야생형 CYP102A1(서열번호 1)의
아미노산 치환 부위of wild-type CYP102A1 (SEQ ID NO: 1)
amino acid substitution site 서열번호Sequence number M16V3M16V3 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 11
(도 8)11
(Fig. 8) G1G1 R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/870N/I881V/E887G/P894S/S954N/M967V/Q981R/A1008D/H1021Y/Q1022E R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A/ A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/870N/MI881V/E887G/P894V/E887G/P894V Q981R/A1008D/H1021Y/Q1022E 12
(도 9)12
(Fig. 9) M179M179 R47L/F81I/F87V/E143G/L188Q/N213S/E267VA474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/ A1008D/H1021Y/Q1022E R47L/F81I/F87V/E143G/L188Q/N213S/E267V A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870QN/I881V/E887G/P894S/S954N/P894S/S954N /Q1022E 13
(도 10)13
(Fig. 10) M221M221 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 F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R/ A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E870N/I881V/E887G/P894S/S954N/M A1008D/H1021Y/Q1022E 14
(도 11)14
(Fig. 11)

* 돌연변이체에 있어 밑줄 친 부분은 야생형 CYP102A1의 환원효소 영역(reductase domain)을 돌연변이 시킴.* The underlined part in the mutant 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-glucoside, for one-glucoside piceatannol glucoside, by　P450　BM3. Enzyme Microb Technol. 97:34-42, 2017..

실험예 1. 야생형 CYP102A1 및 이의 돌연변이체에 의한 나린진 DC의 대사산물 생성의 확인Experimental Example 1. Confirmation of metabolite production of naringin DC by wild-type CYP102A1 and mutants thereof

야생형 CYP102A1(BM3 wt) 및 이의 돌연변이체들(M16V3, G1, M179, M221)이 나린진 DC를 산화시켜 3'-하이드록시화 할 수 있는지 확인하였다. It was confirmed that wild-type CYP102A1 (BM3 wt) and its mutants (M16V3, G1, M179, M221) can oxidize naringin DC to 3'-hydroxylation.

100 mM 인산칼륨 완충액(pH 7.4) 0.25 ml에 각각 야생형 CYP102A1 및 이의 돌연변이체들 100 pmol과 나린진 DC(최종농도 200 μM)을 넣어 전형적인 정상 상태(steady-state) 반응을 수행하였다. In 0.25 ml of 100 mM potassium phosphate buffer (pH 7.4), 100 pmol of wild-type CYP102A1 and its mutants, respectively, and Naringin DC (final concentration 200 μM) were added, and a typical steady-state reaction was performed.

반응을 시작하기 위하여 NADPH-생성 시스템(최종 농도: 1 ml 당 10 mM glucose 6-phosphate, 0.5 mM NADP+ 및 1 IU yeast glucose 6-phosphate dehydrogenase)을 첨가하였다. 메탄올을 이용하여 20 mM 나린진 DC 용액을 제조하고, 효소 반응액으로 희석하여 최종 유기용매 농도가 1%(v/v) 이하가 되도록 하였다. To start the reaction, a NADPH-generating system (final concentration: 10 mM glucose 6-phosphate per 1 ml, 0.5 mM NADP+ and 1 IU yeast glucose 6-phosphate dehydrogenase) was added. A 20 mM naringin DC solution was prepared using methanol and diluted with an enzyme reaction solution so that the final organic solvent concentration was 1% (v/v) or less.

반응액을 37 ℃에서 60 분간 반응시키고, 얼음으로 빙냉시킨 에틸아세테이트 0.6 ml로 반응을 종결시켰다. The reaction solution was reacted at 37° C. for 60 minutes, and the reaction was terminated with 0.6 ml of ice-cooled ethyl acetate.

실험 결과, 야생형 CYP102A1의 돌연변이체들(M16V3, G1, M179, M221)이 모두 높은 활성(1 nmol product/min/nmol P450 이상)으로 나린진 DC의 대사산물을 생성하는 것을 확인할 수 있었다(도 1). As a result of the experiment, it was confirmed that all mutants of wild-type CYP102A1 (M16V3, G1, M179, M221) produced metabolites of naringin DC with high activity (1 nmol product/min/nmol P450 or more) (FIG. 1) .

실험예 2. HPLC 분석 Experimental Example 2. HPLC analysis

상기 실험예 1의 반응혼합물을 원심분리한 후, 유기용매 층 180 μl를 분리하여 질소가스 하에서 증발시키고 HPLC로 분석하였다. 시료(30 μl)를 Gemini C18 컬럼(4.6 mm x 150 mm, 5 μm, 페노메넥스(Phenomenex), 미국)에 주입하였다. 이동상 A는 0.1 % 포름산, 0.5 % 메탄올을 함유하는 물, 이동상 B는 100% 아세토니트릴을 사용하였다. 이동상 A/B(60/40, v/v)를 그래디언트 펌프(LC-20AD, 시마즈(Shimadzu), 일본)를 사용하여 1 ml/min의 속도로 흘려주었다. 용출액을 285 nm의 UV로 측정하였다.After centrifuging the reaction mixture of Experimental Example 1, 180 μl of the organic solvent layer was separated, evaporated under nitrogen gas, and analyzed by HPLC. Samples (30 μl) were injected onto a Gemini C18 column (4.6 mm×150 mm, 5 μm, Phenomenex, USA). Mobile phase A was water containing 0.1% formic acid and 0.5% methanol, and mobile phase B was 100% acetonitrile. Mobile phase A/B (60/40, v/v) was flowed at a rate of 1 ml/min using a gradient pump (LC-20AD, Shimadzu, Japan). The eluate was measured with UV at 285 nm.

CYP102A1이 나린진 DC를 산화시킬 수 있는지 조사하기 위하여, 야생형 CYP102A1(BM3 wt) 및 이의 돌연변이체들(M16V3, G1, M179, M221)을 이용하여 나린진 DC의 산화능을 기질의 농도를 200 μM로 고정시키고 측정하였다. To investigate whether CYP102A1 can oxidize naringin DC, wild-type CYP102A1 (BM3 wt) and its mutants (M16V3, G1, M179, M221) were used to fix the oxidative capacity of naringin DC at a substrate concentration of 200 μM, and measured.

각각 사람 CYP1A2, 박테리아 야생형 CYP102A1 및 이의 돌연변이체들에 의하여 생성된 나린진 DC의 대사산물을 HPLC 크로마토그램(285 nm에서의 UV 흡광도를 측정)을 이용하여 조사하였다.Metabolites of naringin DC produced by human CYP1A2, bacterial wild-type CYP102A1 and mutants thereof, respectively, were investigated using HPLC chromatograms (measured by UV absorbance at 285 nm).

실험 결과, CYP102A1 돌연변이체에 의해 하나의 주요 대사물질이 생성되었다. 상기 주된 대사물질의 화학적 구조를 LC-MS와 NMR 분석을 통해 분석한 결과, 네오에리오시트린 DC임을 확인할 수 있었다(도 2). As a result of the experiment, one major metabolite was produced by the CYP102A1 mutant. As a result of analyzing the chemical structure of the main metabolite through LC-MS and NMR analysis, it was confirmed that it was neoeriocitrin DC (FIG. 2).

실험예 3. LC-MS 분석 및 NMR 분석Experimental Example 3. LC-MS analysis and NMR analysis

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

CYP102A1 돌연변이체 M221을 100 μM의 나린진 DC와 NADPH-생성 시스템의 존재 하에 37 ℃에서 50 분간 반응시켰다. 얼음으로 식힌 2 배의 에틸아세테이트를 가하여 반응을 종결시켰다. 용액을 원심분리한 후, 유기용매층을 분리하여 질소 가스 하에 건조하였다. 반응물을 200 μl 이동상에 볼텍스 믹싱으로 재구성시키고 20 초간 소니케이션 하였다. 제조된 용액의 적정량(10 μl)을 LC 컬럼에 주입하였다.CYP102A1 mutant M221 was reacted with 100 μM of naringin DCs in the presence of a NADPH-generating system at 37° C. for 50 minutes. The reaction was terminated by adding 2 times the amount of ethyl acetate cooled with ice. After centrifuging the solution, the organic solvent layer was separated and dried under nitrogen gas. Reactions were reconstituted by vortex mixing in 200 μl mobile phase and sonicated for 20 s. An appropriate amount (10 μl) of the prepared solution was injected into the LC column.

LC-MS 분석은 LCMS 소프트웨어를 장착한 시마즈 LCMS-2010 EV 시스템을 사용하여 전기분무 이온화 모드(positive)에서 실시하였다. Shim-pack VP-ODS 컬럼(250mm x 2.0mm i.d.; 시마즈)에서 0.1% 트리플루오로아세트산을 함유하는 물과 아세토니트릴(70:30, v/v)로 0.2 ml/min의 유속으로 분리하였다. 대사산물을 확인하기 위하여, 질량 스펙트럼을 전기분무 이온화 모드(positive) 및 SIM 모드에서 기록하였다. 인터페이스와 디텍터 볼트는 각각 4.4 kV 및 1.5 kV이었다. 네블라이제이션 가스 속도는 1.5 ml/min으로 설정하였으며, 인터페이스, curve desolvation line(CDL) 및 히트블록 온도는 각각 250 ℃, 230 ℃ 및 200 ℃로 하였다.LC-MS analysis was performed in electrospray ionization mode (positive) using a Shimadzu LCMS-2010 EV system equipped with LCMS software. Separation was performed on a Shim-pack VP-ODS column (250 mm x 2.0 mm i.d.; Shimadzu) with water containing 0.1% trifluoroacetic acid and acetonitrile (70:30, v/v) at a flow rate of 0.2 ml/min. To identify metabolites, mass spectra were recorded in electrospray ionization mode (positive) and SIM mode. The interface and detector volts were 4.4 kV and 1.5 kV, respectively. The nebulization gas rate was set to 1.5 ml/min, and the interface, curve desolvation line (CDL) and heat block temperatures were set at 250 °C, 230 °C and 200 °C, respectively.

실험 결과, 반응 샘플의 질량 스펙트럼은 18.2 min(네오에리오시트린 DC), 21.1 min(나린진 DC)에서 피크를 나타냈다(도 3). CYP102A1 돌연변이체(M221)에 의해 생성된 대사체 및 기질인 나린진 DC의 질량 스펙트럼은 [M+H]+로 계산하였을 때 각각 582 및 598로 관찰되었다. 이러한 반응혼합물의 LC-MS 분석에 의하여, CYP102A1 돌연변이의 키메라에 의한 네오에리오시트린 DC의 생산을 확인할 수 있었다. As a result of the experiment, the mass spectrum of the reaction sample showed peaks at 18.2 min (Neoeriocitrin DC) and 21.1 min (Naringin DC) ( FIG. 3 ). The mass spectra of the metabolite and substrate naringin DC produced by the CYP102A1 mutant (M221) were observed to be 582 and 598, respectively, when calculated as [M+H] + . By LC-MS analysis of this reaction mixture, it was possible to confirm the production of neoeriocitrin DC by the chimera of the CYP102A1 mutant.

또한, 박테리아 CYP102A1 돌연변이체 M221에 의하여 생성된 대사산물의 화학구조를 NMR 분석 기법을 통하여 분석하였다. NMR 분석은 Varian VNMRS 600 MHz NMR spectrometer 장비를 이용하여 수행되었다. In addition, the chemical structure of the metabolite produced by the bacterial CYP102A1 mutant M221 was analyzed through NMR analysis. NMR analysis was performed using a Varian VNMRS 600 MHz NMR spectrometer.

실험 결과, 상기 생산된 대사산물이 네오에리오시트린 DC인 것을 최종 확인할 수 있었다(도 4). As a result of the experiment, it was finally confirmed that the produced metabolite was neoeriocitrin DC (FIG. 4).

실험예 4. 분자촉매 활성의 결정Experimental Example 4. Determination of Molecular Catalytic Activity

야생형 CYP102A1의 돌연변이체들(M16V3, G1, M179, M221)에 의한 나린진 DC 산화물의 생성속도를 알아보기 위하여, 200 μM의 나린진 DC를 사용한 반응에서 분자촉매활성(turnover number)을 결정하였다. 나린진 DC의 생성속도의 측정을 위해 상기 실험예 2에 기재한 바와 같은 HPLC를 수행하였다. 구체적으로, 나린진 DC의 농도를 10 μM에서 500 μM까지 준비하여 30 분간 반응을 수행하였다. To investigate the production rate of naringin DC oxide by wild-type CYP102A1 mutants (M16V3, G1, M179, M221), molecular catalytic activity (turnover number) was determined in a reaction using 200 μM of naringin DC. In order to measure the rate of naringin DC production, HPLC was performed as described in Experimental Example 2 above. Specifically, the concentration of naringin DC was prepared from 10 μM to 500 μM, and the reaction was performed for 30 minutes.

실험 결과, 나린진 DC의 산화에 따른 대사산물의 생성에 대한 상기 표 4의 4 종의 돌연변이체의 분자촉매활성은 넓은 범위에서 다양하였다. 반면, 야생형 CYP102A1의 촉매활성은 확인할 수 없었다. 야생형 CYP102A1의 돌연변이체 중 M16V3, M179는 낮은 활성을 보였으며, 상기 4종의 돌연변이체중 G1, M221은 다른 돌연변이체에 비해 2~3배 높은 활성을 나타내었다(도 5). As a result of the experiment, the molecular catalytic activity of the four mutants of Table 4 on the production of metabolites according to the oxidation of naringin DC varied in a wide range. On the other hand, the catalytic activity of wild-type CYP102A1 could not be confirmed. Among the wild-type CYP102A1 mutants, M16V3 and M179 showed low activity, and among the four mutants, G1 and M221 showed 2-3 times higher activity than other mutants ( FIG. 5 ).

CYP102A1 돌연변이체 4종류(M16V3, G1, M179, M221)의 효소를 선택하여 대사산물을 생성하는 효율을 속도록적 매개변수를 이용하여 비교하여 하기 표 5에 나타내었다:Table 5 below shows the efficiency of selecting four types of CYP102A1 mutant enzymes (M16V3, G1, M179, and M221) to produce metabolites and comparing them using fast-acting parameters:

CYP102A1CYP102A1 네오에리오시트린 DC의 생성Generation of neoeriocitrin DC 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 ) M16V3M16V3 7.8 ± 0.57.8 ± 0.5 160 ± 25160 ± 25 0.048 ± 0.0200.048 ± 0.020 G1G1 11.0 ± 0.311.0 ± 0.3 73 ± 673 ± 6 0.150 ± 0.0500.150 ± 0.050 M179M179 5.3 ± 0.25.3 ± 0.2 76 ± 976 ± 9 0.070 ± 0.0220.070 ± 0.022 M221M221 10.8 ± 0.210.8 ± 0.2 79 ± 679 ± 6 0.136 ± 0.0330.136 ± 0.033

상기의 결과들로부터 박테리아 CYP102A1 돌연변이 효소들이 네오에리오시트린 DC 산물을 주요 대사산물로 생성한다는 것을 확인할 수 있었다.From the above results, it was confirmed that the bacterial CYP102A1 mutant enzymes produced neoeriocitrin DC products as major metabolites.

감귤류에 다량 존재하는 나린진 DC의 산화는 박테리아 CYP102A1의 돌연변이체에 의하여 촉매되며, 주요한 대사산물로 히드록실화 산물, 즉, 네오에리오시트린 DC가 히드록실화 반응에 의하여 생성됨을 확인하였다. It was confirmed that the oxidation of naringin DC, which is abundantly present in citrus fruits, is catalyzed by the mutant of bacterial CYP102A1, and that the hydroxylation product, that is, neoeriocitrin DC, is produced by the hydroxylation reaction as a major metabolite.

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 only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also 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 claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for preparing neoeriocitrin dihydrochalcone <130> P-190127 <160> 14 <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> BamH I forward primer <400> 3 agcggatcca tgacaattaa agaaatgcct c 31 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sac I Reverse primer <400> 4 atcgagctcg tagtttgtat 20 <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 M16V3 <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 G1 <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 Ala 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 Arg Gln Phe Ala Leu His Glu Ala Thr Leu Ala 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 M179 <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 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> 14 <211> 1048 <212> PRT <213> Artificial Sequence <220> <223> CYP102A1 mutant M221 <400> 14 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 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for preparing neoeriocitrin dihydrochalcone <130> P-190127 <160> 14 <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 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 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 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 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> BamH I forward primer <400> 3 agcggatcca tgacaattaa agaaatgcct c 31 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sac I Reverse primer <400> 4 atcgagctcg tagtttgtat 20 <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 M16V3 <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 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 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 G1 <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 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 Ala 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 Arg Gln Phe Ala Leu His Glu Ala Thr Leu Ala 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 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 M179 <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 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 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 M221 <400> 14 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 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 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 네오에리오시트린 디하이드로칼콘(neoeriocitrin dihydrochalcone) 제조용 조성물로서,
상기 CYP102A1는 서열번호 1의 아미노산 서열로 이루어진 것을 특징으로 하고,
상기 CYP102A1의 돌연변이체는 상기 서열번호 1로부터 치환되는 아미노산이 R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V/E143G/L188Q/N213S/E267V 및 F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R으로 구성되는 군으로부터 선택되는 하나 이상인 것을 특징으로 하며,
상기 효소의 반응 기질은 나란진 디하이드로칼콘인 것을 특징으로 하는 네오에리오시트린 디하이드로칼콘 제조용 조성물.
A composition for preparing neoeriocitrin dihydrochalcone comprising one or more enzymes selected from the group consisting of mutants of CYP102A1,
The CYP102A1 is characterized in that it consists of the amino acid sequence of SEQ ID NO: 1,
The mutant of CYP102A1 has amino acids substituted from SEQ ID NO: 1 R47L/F81I/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/T152A/L188Q/E267V/Q403R/V413A, R47L/F81I/F87V It is characterized in that at least one selected from the group consisting of /E143G/L188Q/N213S/E267V and F11Y/R47L/F81I/F87V/E143G/L188Q/E267V/H408R,
The reaction substrate of the enzyme is a composition for preparing neoeocitrin dihydrochalcone, characterized in that naranjin dihydrochalcone.
 제 1 항에 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/Q1022E인 것을 추가로 포함하는 것을 특징으로 하는 네오에리오시트린 디하이드로칼콘 제조용 조성물.
The method of claim 1,
The mutant of CYP102A1 has an amino acid substituted from SEQ ID NO: 1 A474V/E558D/T664A/P675L/A678E/E687A/A741G/K813E/R825S/R836H/E870N/I881V/E887G/P894S/S954N/M967V/Q981R/A8D /H1021Y/Q1022E Neo eriocitrin dihydrochalcone preparation composition, characterized in that it further comprises.
 제 1 항 또는 제 2 항의 네오에리오시트린 디하이드로칼콘 제조용 조성물을 포함하는 네오에리오시트린 디하이드로칼콘의 생산용 키트.
A kit for the production of neo eriocitrin dihydrochalcone comprising the composition of claim 1 or 2 for preparing the neoeocitrin dihydrochalcone.
 제 1 항 또는 제 2 항의 네오에리오시트린 디하이드로칼콘 제조용 조성물과 나린진 디하이드로칼콘을 반응시키는 단계를 포함하는 네오에리오시트린 디하이드로칼콘의 제조방법.
[Claim 3] A method for producing neo-eryocitrin dihydrochalcone comprising reacting the composition for preparing neo-eryocitrin dihydrochalcone according to claim 1 or 2 with naringin dihydrochalcone.
 제 4 항에 있어서,
NADPH-생성 시스템을 추가하는 단계를 더 포함하는 것을 특징으로 네오에리오시트린 디하이드로칼콘의 제조방법. The method of claim 4,
A method for producing neoeriocitrin dihydrochalcone, characterized in that it further comprises adding a NADPH-generating system.
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Biosci. Biotechnol. Biochem., Vol. 77, pp. 1340-1343 (2013.06.07.)
Biotechnol. Bioeng., Vol. 111, pp. 1313-1322 (2014.)
Microb. Cell Fact., Vol. 15, pp. 135 (1-15) (2016.08.05.)
NCBI GenBank Accession No. ADA57059.1 (2011.03.25.)
Trends in Biotechnology, Vol. 25, pp. 289-298 (2007.)

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