KR0184716B1 - Bacillus sp. lk-2 having d-alanine aminotransferase - Google Patents

Bacillus sp. lk-2 having d-alanine aminotransferase Download PDF

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KR0184716B1
KR0184716B1 KR1019960000709A KR19960000709A KR0184716B1 KR 0184716 B1 KR0184716 B1 KR 0184716B1 KR 1019960000709 A KR1019960000709 A KR 1019960000709A KR 19960000709 A KR19960000709 A KR 19960000709A KR 0184716 B1 KR0184716 B1 KR 0184716B1
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전영중
정상철
성문희
이승구
홍승표
노현수
곽미선
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제일제당주식회사
김은영
한국과학기술연구원
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Abstract

본 발명은 D-아미노산의 아미노기를 케토산에 전이시키는 내열성 D-알라닌 아미노기전이효소 (D-alanine aminotransferase)를 갖는 신규의 고온성 균주 바실러스 종 LK-2(기탁번호: KCTC 8717P)에 관한 것이다.The present invention relates to a novel high temperature strain Bacillus sp. LK-2 (Accession No .: KCTC 8717P) having a heat-resistant D-alanine aminotransferase which transfers the amino group of D-amino acid to keto acid.

Description

내열성 D-알라닌 아미노기전이효소 활성을 갖는 신규의 고온성 균주 바실러스 종 LK-2Novel thermophilic strain Bacillus sp. LK-2 with heat-resistant D-alanine aminotransferase activity

제1도는 고온성 균주 바실러스(Bacillus) 종 LK-2 (기탁번호: KCTC 8717P)가 생산하는 내열성 D-알라닌 아미노기전이효소의 활성에 대한 반응온도의 영향을 나타내는 그래프이다.1 is a graph showing the effect of reaction temperature on the activity of the heat-resistant D-alanine aminotransferase produced by the pyrogenic strain Bacillus sp. LK-2 (Accession No .: KCTC 8717P).

본 발명은 내열성 D-알라닌 아미노기전이효소 활성을 갖는 신규의 고온성 균주 바실러스 종 LK-2(Bacillus sp. LK-2)에 관한 것이다. 좀더 구체적으로, 본 발명은 D-아미노산의 아미노기를 케토산에 전이시키는 내열성 D-알라닌 아미노기전이효소(D-alanine aminotransferase) 활성을 갖는 신규의 고온성 균주 바실러스 종 LK-2 에 관한 것이다.The present invention relates to a novel high temperature strain Bacillus sp. LK-2 having heat resistant D-alanine aminotransferase activity. More specifically, the present invention relates to a novel thermophilic strain Bacillus sp. LK-2 having heat-resistant D-alanine aminotransferase activity that transfers the amino group of D-amino acid to keto acid.

자연계에 존재하는 아미노산들은 대부분 광학활성을 나타내는 비대칭탄소를 함유하고 있는데, 이들은 보통 L-구조 상태로 존재하며, 예를들어 미생물 세포벽을 구성하는 아미노산과 같은 소수의 아미노산들 만이 자연상태에서 D-구조를 나타낸다.Most amino acids in nature contain asymmetric carbons, which are optically active. They usually exist in the L-structure and only a few amino acids, such as amino acids that make up the microbial cell wall, are naturally D-structured. Indicates.

오늘날 D-아미노산은 합성감미료, β-락탐계 반합성항생제, 펩티드 호르몬, 살충제 등을 생산하기 위한 중간물질로서 식품 및 의약분야에 널리 이용되고 있으며, 특히 효소분해에 대해 보다 안정한 생리활성물질을 생산하기 위해 D-아미노산으로 어떤 물질의 말단을 보호함으로써 L-이성체에 특이적인 활성을 갖는 분해효소가 기질을 인식하지 못하도록 하는 용도로도 개발되는 등 점차 그 용도가 다양해지고 있다. 그러나, 이와 같이 다양한 용도를 갖는 D-아미노산을 화학적 합성법으로 제조하고자 하면 반응 후 언제나 D, L-아미노산의 혼합물 상태로 얻어지기 때문에 까다로운 광학적 순수 정제과정을 거쳐야 하는 등의 어려운 점이 있다.Today, D-amino acids are widely used in food and medicine as intermediates for the production of synthetic sweeteners, β-lactam antisynthetic antibiotics, peptide hormones, pesticides, etc., especially to produce more stable bioactive substances against enzymatic degradation. In order to protect the ends of certain substances with D-amino acids, their use is gradually diversified, such that they are developed for the purpose of preventing degradation of enzymes having L-isomer-specific activity. However, when the D-amino acid having various uses is prepared by a chemical synthesis method, it is difficult to undergo a difficult optical pure purification process because it is always obtained as a mixture of D and L-amino acids after the reaction.

화학적 합성법에 상기한 바와 같은 문제점이 있으므로 천연의 D-아미노산 아미노기전이효소를 이용하여 D-아미노산을 합성하는 방법이 모색되었다.Since the chemical synthesis has the problems described above, a method of synthesizing D-amino acid using natural D-amino acid aminotransferase has been sought.

아미노기전이효소는 생체내 아미노산의 대사에 있어 한 아미노산을 다른 아미노산으로 전환시키는 작용을 하는 것으로서 피리독살(pyridoxal) 또는 피리독사민(pyri-doxamine)의 인산염 및 알파-케토산으로 부터 새로운 아미노산을 생성시킨다.Aminotransferases convert amino acids into other amino acids in the metabolism of amino acids in vivo, producing new amino acids from phosphates and alpha-keto acids of pyridoxal or pyri-doxamine. Let's do it.

특히, 자연상태의 D-아미노산 아미노기전이효소로는 미생물 세포벽의 펩티도글리칸을 구성하는 D-아미노산 합성 효소를 언급할 수 있는데, 이들은 공지의 방법(참조: Jones, W. et al., (1985) Methods Enzymol. 113, 108-113)에 의하여 취득가능하다.In particular, as a natural D-amino acid aminotransferase, mention may be made of D-amino acid synthetase constituting peptidoglycan of microbial cell walls, which are known methods (Jones, W. et al., ( 1985) Methods Enzymol. 113, 108-113).

한편, 고온균으로부터의 D-알라닌 아미노기전이효소(D-alanine aminotransferase: EC 2.6.1.21) 탐색은 일본의 타니자와 등(참조: Tanizawa et al., (1989)J. Biol. Chem. 264, 2445-2449)에 의하여 이루어졌으며, 최근에는 효소의 정확한 반응기작 및 단백질 구조에 관한 연구결과가 보고되었다(참조: Sugio et al., (1995) Biochemistry, 34, 9661-9669). D-알라닌 아미노기전이효소의 반응기작은 먼저 기질인 D-알라닌의 아미노기를 효소의 피리독신-5'-인산에 전이시킴으로써 이에 상응하는 피루빈산 및 피리독사민-5'-인산을 생성하는 반쪽반응(half reaction)과 새로운 기질인 α-케토글루타르산이 피리독사민-5'-인산으로 부터 아미노기를 받아들여서 D-글루탐산을 생성하는 반쪽반응으로 구성되어 있다.On the other hand, the search for D-alanine aminotransferase (EC 2.6.1.21) from pyrobacterial cells was carried out in Tanizawa et al. (Tanizawa et al., (1989) J. Biol. Chem. 264, Japan). 2445-2449), and recent studies on the exact reaction mechanism and protein structure of enzymes have been reported (Sugio et al., (1995) Biochemistry, 34, 9661-9669). The reaction mechanism of the D-alanine aminotransferase first involves the transfer of the amino group of D-alanine, a substrate, to the pyridoxine-5'-phosphate of the enzyme to produce the corresponding pyruvic acid and pyridoxamine-5'-phosphate. half reaction) and a new substrate, α-ketoglutaric acid, are half-reactions that take in amino groups from pyridoxamine-5'-phosphate to produce D-glutamic acid.

효소를 생물촉매로 이용하는 생물공정에 있어서 가장 중요한 것은 효소의, 즉 생물촉매의 안정성이다. D-알라닌 아미노기전이효소의 효소반응을 이용하여 각종 고부가가치의 유용한 D-아미노산을 합성 및 생산하기 위한 효소공정에 있어서도 생물촉매의 안정성은 생산성의 증대에 있어서 매우 중요한 요인중 하나이다. 일반적으로 고온의 환경에 적응하여 증식하는 고온성 미생물은 환경의 영향에 의해 열에 대하여 안정한 내열성 효소를 가지고 있다고 알려져 있다. 그리고, 이러한 내열성효소는 열에 안정한 특징 이외에도 유기용매에 대한 안정성, 극단의 수소이온 농도에 대한 안정성 및 화학변성체에 대한 안정성도 가지고 있다고 알려져 있다. 따라서, 고온성 미생물이 가지고 있는 내열성 D-아미노산 아미노기전이효소는 상기에서 언급한 효소안정성을 획득하고 있으므로 각종의 고부가가치 유용 D-아미노산의 합성 및 생산기술개발에 생물촉매로서 매우 유용하게 사용될 것으로 생각된다.The most important thing in the bioprocess using enzymes as biocatalysts is the stability of the enzymes, ie biocatalysts. In the enzymatic process for synthesizing and producing various high value-added useful D-amino acids using enzymatic reaction of D-alanine aminotransferase, the stability of biocatalyst is one of the important factors in the increase of productivity. Generally, it is known that thermophilic microorganisms that adapt to a high temperature environment and have a heat-resistant enzyme that is stable to heat by environmental influences. In addition to the heat-stable enzymes, these heat-resistant enzymes are known to have stability against organic solvents, extreme hydrogen ion concentrations, and chemically modified substances. Therefore, the heat-resistant D-amino acid aminotransferase possessed by the thermophilic microorganisms has obtained the enzyme stability mentioned above, and thus it is considered to be very useful as a biocatalyst for the synthesis and production of various high value-added useful D-amino acids. do.

이에 본 발명자들은 국내 각지의 고온균 서식지로 부터, 열에 안정하여 고온에서의 효소반응이 가능할 뿐 아니라 D-알라닌을 비롯하여 D-발린 등의 분지 아미노산에도 높은 반응성을 나타내는 새로운 기질특이성의 D-알라닌 아미노기전이효소 생산 미생물의 탐색을 목표로 집중적인 연구를 수행하였으며, 그 결과 천여종의 고온성 미생물로 부터 D-알라닌 및 D-발린 등의 합성에 가장 적합한 D-알라닌 아미노기전이효소를 보유하고 있는 것으로 판단된 1종의 미생물을 순수하게 분리하여 본 발명을 완성하게 되었다. 또한, 본 발명의 고온성 미생물을 동정한 결과 기존의 바실러스 스패리쿠스(Bacillus sphaericus)와 매우 유사한 생리적 특성을 갖는 한편, 본 발명자들에 의해 대한민국 특허 출원 제 95-53557호로 출원된 바실러스 LK-1 균주와는 상이한 몇가지 특성을 나타내었으므로 본 발명자들은 이 고온성 미생물을 바실러스 종 LK-2로 명명하였다.Therefore, the present inventors are not only capable of thermally stable enzymatic reaction at high temperature from the habitats of high temperature bacteria in Korea, but also have a new substrate-specific D-alanine amino group showing high reactivity to branched amino acids such as D-alanine and D-valine. Intensive research aimed at the search for transferase-producing microorganisms, and as a result, it was found that it possesses D-alanine aminotransferase, which is most suitable for the synthesis of D-alanine and D-valine from a thousand kinds of thermophilic microorganisms. One kind of microorganisms was isolated to complete the present invention. In addition, as a result of identifying the thermophilic microorganism of the present invention, while having physiological characteristics very similar to the existing Bacillus sphaericus, Bacillus LK-1 filed by the present inventors in Korean Patent Application No. 95-53557 The thermophilic microorganisms were termed Bacillus sp. LK-2 because they exhibited several different characteristics from the strain.

이하, 본 발명의 구성을 상세히 설명한다.Hereinafter, the configuration of the present invention will be described in detail.

본 발명은 D-아미노산의 아미노기를 케토산에 전이시키는 내열성 D-알라닌 아미노기전이효소 활성을 갖는 고온성 균주 바실러스 종 LK-2에 관한 것이다. 본 발명에 따른 바실러스 종 LK-2 균주를 분리하고 동정한 과정은 다음과 같다.The present invention relates to a high temperature strain Bacillus sp. LK-2 having a heat resistant D-alanine aminotransferase activity for transferring the amino group of D-amino acid to keto acid. The process of isolating and identifying Bacillus sp. LK-2 strain according to the present invention is as follows.

미생물의 분리: 먼저 전국 각지의 퇴비제조토양, 부식토, 하천, 폐수, 온천 등지에서 채취한 샘플을 MY 배지(1.5% 폴리펩톤(polypeptone), 0.2% 효모추출물(yeast extract), 0.2% 고기추출물(meat extract), 0.2% 글리세롤, 0.2% K2HPO4, 0.2%KH2PO4, 0.026% NH4Cl 로 구성)에 접종하고 55 내지 60℃ 에서 부유배양(enrichment culture)하였다. 이 배양액을 2% 아가(agar)를 포함한 MY 평판배지(agar plate medium)에 접종하고 55 내지 60℃에서 배양하여 순수한 콜로니를 분리하였다. 분리된 균주를 5㎖ MY 액체배지에서 8 내지 10시간 배양한 후, 균체를 수확하여 초음파파쇄기로 균체를 파쇄하여 조효소액으로 만든 다음 D-알라닌 아미노기전이효소 활성을 측정하였다.Microorganism Separation: First, samples from compost manufacturing soils, humus soils, rivers, wastewaters, and hot springs are collected from MY media (1.5% polypeptone, 0.2% yeast extract, 0.2% meat extract). meat extract), 0.2% glycerol, 0.2% K 2 HPO 4 , 0.2% KH 2 PO 4 , 0.026% NH 4 Cl) and incubated at 55-60 ° C. (enrichment culture). The culture was inoculated in MY plate medium containing 2% agar (agar) and cultured at 55 to 60 ℃ pure colony was isolated. After the isolated strain was incubated in 5 ml MY liquid medium for 8 to 10 hours, the cells were harvested, and the cells were crushed with an ultrasonic crusher to make a coenzyme solution, and then D-alanine aminotransferase activity was measured.

D-알라닌 아미노기전이효소 활성을 측정하기 위한 반응액은 0.1M 트리스완충액(Tris-HCl, pH 8.5)0.2㎖ 에 각각 10mM 의 D-알라닌, α-케토글루타르산과 배양액 0.1㎖에 해당하는 조효소액을 포함하도록 조제되었으며, 50℃에서 1시간 동안 정치 반응시켰다.The reaction solution for measuring D-alanine aminotransferase activity was 0.2 ml of 0.1 M Tris buffer solution (Tris-HCl, pH 8.5), and 10 mM D-alanine, α-ketoglutaric acid and coenzyme solution corresponding to 0.1 ml of culture medium. It was prepared to include, and allowed to react for 1 hour at 50 ℃.

D-알라닌 아미노기전이효소의 반응에 의하여 생성된 피루베이트의 양은 반응액에 60% 수산화칼륨염 용액 0.2㎖ 및 2% 살리실알데히드(salicylaldehyde)용액 0.1㎖를 가한 후 37℃에서 30분간 정치반응시켜 발색시킨 다음, 물로 2배 희석하고 480nm 에서 흡광도를 측정하는 방법으로 결정하였다(참조 : Berntsson S., 1995, Anal.,chem., 27, 1659-1660).The amount of pyruvate produced by the reaction of D-alanine aminotransferase was added to the reaction solution with 0.2 ml of 60% potassium hydroxide solution and 0.1 ml of 2% salicylaldehyde solution, followed by stationary reaction at 37 ° C for 30 minutes. Color development was followed by dilution with water twice and measurement of absorbance at 480 nm (Berntsson S., 1995, Anal., Chem., 27, 1659-1660).

1차 균주 선별과정에서 D-알라닌 아미노기전이효소 활성을 나타낸 미생물 100여종에서 D-알라닌 아미노기전이효소의 활성이 비교적 높은 4종의 고온성 미생물은 하기 표 1에 나타낸 바와 같다.Four high-temperature microorganisms having relatively high activity of D-alanine aminotransferase in about 100 microorganisms showing D-alanine aminotransferase activity in the first strain selection process are shown in Table 1 below.

상기 분리된 4종의 고온성 미생물에 대해서 분지아미노산인 D-발린 합성에 대한 활성이 높은 미생물을 재선별하기 위한 실험은 다음과 같이 수행하였다.The experiment for reselecting the microorganisms having high activity on the synthesis of branched amino acid D-valine for the four high-temperature microorganisms isolated was performed as follows.

D-발린을 합성하기 위한 반응액은 0.05M 인산염완충액(pH 8.5) 0.2㎖에 각각 10mM 의 D-글루탐산, α-케토이소발레르산과 배양액 0.2㎖에 해당하는 조효소액을 포함하도록 조제되었으며, 50℃ 에서 1시간동안 정치반응을 수행한 후, 광학활성 TLC(chiral TCL)분석을 통하여 생성된 D-발린을 비교하였다. 광학활성 TLC는 아세토니트릴(acetonitrile)/메탄올/물을 4/1/1의 부피비로 포함한 용매를 사용하여 20분동안 전개한 후, 2% 닌히드린(ninhidrin)으로 발색시키는 방법을 사용하였다. 그 결과, 분리된 고온성 미생물중 417번 미생물이 다른 것들에 비하여 D-발린에 대해 비교적 높은 아미노기전이효소 활성을 나타내었으므로 이를 다양한 분지 아미노산의 생산에 적합한 미생물로 선택하고 이 미생물의 동정을 하기와 같이 수행하였다.The reaction solution for synthesizing D-valine was prepared to contain 0.2 ml of 0.05M phosphate buffer (pH 8.5) and 10 mM D-glutamic acid, α-ketoisovaleric acid and coenzyme solution corresponding to 0.2 ml of the culture medium. After stationary reaction for 1 hour at ℃, D- valine produced by optically active TLC (chiral TCL) analysis was compared. Optically active TLC was developed using a solvent containing acetonitrile / methanol / water in a volume ratio of 4/1/1 for 20 minutes and then developing with 2% ninhidrin. As a result, microorganism No. 417 of the isolated thermophilic microorganisms exhibited relatively high aminotransferase activity against D-valine compared to others, and thus, it was selected as a microorganism suitable for the production of various branched amino acids and identified. Was carried out as follows.

미생물의 동정 : 상기와 같이 분리된 고온성 미생물은 417은 2% 아가평판배지에서 배양하였을 때 크림색의 반투명한 콜로니를 형성하였다. 이 미생물은 30℃ 이상 60℃ 이하의 온도 및 호기조건에서 생육가능하지만, 30℃ 에서는 약 48시간이 지난 후에 자라기 시작하였으며 65℃에서는 전혀 자라지 못하여 중등도 고온성 미생물(moderate thermophile)에 속하는 것으로 나타났다. 또한, 그림양성이고 내생포자를 형성하며, 현미경으로 관찰하면 55℃의 호기조건에서 분리된 다른 균들에 비하여 길이 방향으로 길게 성장하는 특징이 있는 간균이었다.Identification of microorganisms: The high-temperature microorganisms isolated as described above formed 417 translucent colonies when cultured in 2% agar plate medium. The microorganisms were able to grow at temperatures above 30 ° C. and 60 ° C., but they began to grow after about 48 hours at 30 ° C., but did not grow at 65 ° C., indicating that they belong to moderate thermophile. In addition, it is a picture-positive, endogenous spores, and when observed under a microscope it was characterized by a longer growth in the longitudinal direction than other bacteria isolated under aerobic conditions of 55 ℃.

상기 고온성 미생물의 상세한 생리적 및 생화학적 동정결과는 하기 표2에 나타낸 바와 같다.Detailed physiological and biochemical identification results of the pyrogenic microorganisms are shown in Table 2 below.

이상의 결과로 부터 본 발명에서 새로이 분리된 균주는 포도당을 비롯한 당류를 탄소원으로 이용하지 못하는 특성을 가지는 바실러스 스패리쿠스(Bacillus sphaericus)와 매우 유사한 생리적 특성을 지닌 것으로 나타났다. 또한, 본 발명에 따른 균주는 본 발명자들에 의해 특허출원 제95-53557호로 대한민국 특허청에 1995. 12. 21자 출원된 바실러스 LK-1과 하기에 기술하는 3가지의 중요한 차이를 나타내고 있다 :From the above results, it was shown that the newly isolated strain has a physiological characteristic very similar to Bacillus sphaericus, which has a property of not using glucose or other sugars as a carbon source. In addition, the strain according to the present invention exhibits three important differences as described below with Bacillus LK-1, filed on December 21, 1995, filed with the Republic of Korea Patent Application No. 95-53557 by the inventors:

1) 본 발명의 균주는 바실러스 LK-1에 비하여 길이방향으로 길게 성장하는 특징이 있고, 포자의 크기가 더 크게 관찰되었다.1) The strain of the present invention is characterized by a longer growth in the longitudinal direction than Bacillus LK-1, the spore size was observed larger.

2) 바실러스 LK-1은 배양온도 30℃에서 수시간 후에 자라기 시작하지만 본 발명은 균주는 약 48시간이 지난 후에 생육이 관찰되었다.2) Bacillus LK-1 begins to grow after several hours at the culture temperature 30 ℃ but the present invention was observed after about 48 hours the strain growth.

3) 영양배지(nutrient broth)에서 배양한 본 발명의 균주는 D-아미노산 아미노기전이효소 비활성에 있어서 바실러스 LK-1보다 4 내지 5배 높다.3) The strain of the present invention cultured in nutrient broth is 4-5 times higher than Bacillus LK-1 in D-amino acid aminotransferase inactivation.

따라서, 본 발명에 따른 균주는 기존의 바실러스 LK-1과도 상이한 여러특징을 보일 뿐아니라 D-아미노산 아미노기전이효소의 비활성면에서 월등히 향상된 특성을 나타내므로 신규의 고온성 바실러스 종으로 동정되었으며, 본 발명자들은 이를 바실러스 종 LK-2로 명명하고 1995년 12월 13일자로 한국과학기술연구원 부설 생명공학연구소내 유전자원센타에 기탁완료하고 기탁번호(KCTC 8717P)를 부여받았다.Therefore, the strain according to the present invention was shown to be a novel thermophilic Bacillus species because it not only shows a number of features different from the existing Bacillus LK-1, but also shows significantly improved properties in terms of inactivation of D-amino acid aminotransferase. They named it Bacillus sp. LK-2, and on December 13, 1995, they deposited it at the Genetic Resources Center at the Korea Institute of Science and Technology, and received a deposit number (KCTC 8717P).

효소의 내열성 : 본 발명에 따른 고온성 바실러스 종 LK-2가 생산하는 내열성 D-알라닌 아미노기전이효소의 반응최적온도 및 열안정성을 조사하기 위하여 다음과 같이 실험하였다. 효소반응의 최적온도는 각각 10mM의 D-알라닌과 α-케토글루타르산을 포함한 0.1M 트리스완충액 0.2㎖에 상기 미생물의 조효소액을 가하고 30 내지 70℃까지 온도를 변화시켜가면서 효소반응을 수행함으로써 결정하였다. 그 결과, 제1도에 나타낸 바와 같이 이 효소는 55 내지 60℃의 반응온도에서 최대활성을 나타내었으므로 50℃ 이상의 고온반응에 적합한 내열성 효소인 것을 확인할 수 있었다.Heat resistance of enzyme: In order to investigate the optimum temperature and thermal stability of the heat-resistant D-alanine aminotransferase produced by the high temperature Bacillus sp. LK-2 according to the present invention, the following experiments were performed. The optimum temperature of the enzyme reaction was obtained by adding the coenzyme solution of the microorganism to 0.2 ml of 0.1M tris buffer containing 10 mM D-alanine and α-ketoglutaric acid, respectively, and performing the enzymatic reaction by varying the temperature to 30 to 70 ° C. Decided. As a result, as shown in FIG. 1, the enzyme showed the maximum activity at the reaction temperature of 55 to 60 ℃, it was confirmed that it is a heat-resistant enzyme suitable for high temperature reaction of 50 ℃ or more.

이와 같이, 본 발명에 따른 바실러스 종 LK-2 균주는 D-아미노산의 아미노기를 케토산에 전이시키는 능력을 보유할 뿐아니라 고온의 반응조건하에서도 효소반응이 안정적으로 수행될 수 있으므로 D-알라닌, D-발린 등의 합성에 적합하며 산업적으로도 매우 유용하게 사용될 수 있다.As such, the Bacillus sp. LK-2 strain according to the present invention not only possesses the ability to transfer the amino group of D-amino acid to keto acid, but also can be stably performed under high temperature reaction conditions, so that D-alanine, It is suitable for the synthesis of D-valine and the like and can be very useful industrially.

바실러스 종 LK-2 균주에 의해 아미노기 공여용 기질로 이용될 수 있는 D-아미노산으로는 D-알라닌, D-발린, D-글루탐산 등을 언급할 수 있고, 아미노기를 받는 케토산으로는 피루브산, α-케토글루타르산, α-케토이소발레르산 등을 들 수 있으며 이들을 기질로 사용하는 경우에 각 기질에 상응하는 D-아미노산이 고수율로 제조된다.D-amino acids that may be used as substrates for donating amino groups by the Bacillus sp. LK-2 strain may include D-alanine, D-valine, D-glutamic acid, and the like. Keto acids that receive amino groups include pyruvic acid, α -Ketoglutaric acid, α-ketoisovaleric acid, and the like. When these are used as substrates, D-amino acids corresponding to each substrate are prepared in high yield.

본 발명의 균주로 부터 얻어진 조효소액은 D-아미노산을 제조하는 반응의 촉매로 사용될 수 있으며, 조효소액은 균주의 배양액으로 부터 균체를 수확한 후 파쇄하여 제조한다.The crude enzyme solution obtained from the strain of the present invention can be used as a catalyst for the reaction for producing D-amino acid, and the crude enzyme solution is prepared by harvesting and crushing the cells from the culture medium of the strain.

이하, 본 발명에 따른 균주의 유용성을 하기 참고예에 의거하여 보다 구체적으로 설명한다.Hereinafter, the usefulness of the strain according to the present invention will be described in more detail based on the following reference example.

[참고예 1]Reference Example 1

본 발명에 따른 고온성 바실러스 종 LK-2(기탁번호 : KCTC 8717P)를 펩톤 5g/ℓ 및 고기추출물 3g/ℓ를 함유하는 배양액중에서 8시간동안 배양하여 내열성 D-알라닌 아미노기전이효소를 생산한 후, 세포를 파쇄하고 상등액을 분리하여 D-알라닌 및 α-케토글루타르산을 기질로 사용하는 하기의 D-글루탐산 생산반응에 이용하였다.The thermophilic Bacillus sp. LK-2 (Accession No .: KCTC 8717P) according to the present invention was incubated for 8 hours in a culture solution containing 5 g / l peptone and 3 g / l meat extract to produce a heat-resistant D-alanine aminotransferase. The cells were disrupted and the supernatant was separated and used for the following D-glutamic acid production using D-alanine and α-ketoglutaric acid as substrates.

D-알라닌 및 α-케토글루타르산의 농도가 각각 0.89g/ℓ, 2.26g/ℓ이고 고온성 바실러스 종 LK-2 균주의 조효소액을 단백질 농도 0.25g/ℓ가 되도록 첨가한 0.1M 인산염완충액(pH 8.5)을 반응액으로 사용하였다. 이 반응액을 37℃에서 2시간 동안 반응시킨 후 광학활성 TLC로 분석한 결과, 약 30%의 수율로 D-글루탐산이 생성되었다. 한편, 온도를 50℃로 승온시켜 2시간동안 반응시킨 후 광학활성 TLC로 분석한 결과, 약 50%의 수율로 D-글루탐산이 생성되었으므로 반응온도가 높아짐에 따라 D-글루탐산의 수율이 높아짐을 확인하였다. 또한, 효소의 잔존활성을 측정한 결과 효소 활성이 거의 실활되지 않았음을 확인하였다.0.1M phosphate buffer solution containing 0.89g / l and 2.26g / l of D-alanine and α-ketoglutaric acid, respectively, and coenzyme solution of high temperature Bacillus sp. (pH 8.5) was used as the reaction solution. The reaction solution was reacted at 37 ° C. for 2 hours and analyzed by optically active TLC. As a result, D-glutamic acid was produced in a yield of about 30%. On the other hand, after the temperature was raised to 50 ℃ and reacted for 2 hours and analyzed by optically active TLC, D- glutamic acid was produced in a yield of about 50%, so that the yield of D- glutamic acid increases as the reaction temperature increases It was. In addition, as a result of measuring the remaining activity of the enzyme, it was confirmed that the enzyme activity was almost inactivated.

[참고예 2]Reference Example 2

D-아미노산 및 케토산으로 각각 D-글루탐산 및 α-케토이소발레르산을 1.47g/ℓ, 1.38g/ℓ 농도로 사용하고, 50℃로 고정된 반응온도에서 2시간동안 반응시키는 점을 제외하고는 참고예 1에서와 동일한 방법으로 수행하여 D-발린을 합성하였다. 반응용액을 광학활성 TLC로 분석한 결과 약 20%의 수율로 D-발린이 생성되었음을 확인하였다.D-glutamic acid and α-ketoisovaleric acid were used as D-amino acid and keto acid at a concentration of 1.47 g / l and 1.38 g / l, respectively, except that they were reacted at a reaction temperature fixed at 50 ° C. for 2 hours. Then, D-valine was synthesized in the same manner as in Reference Example 1. Analysis of the reaction solution by optically active TLC confirmed that D-valine was produced in about 20% yield.

Claims (1)

D-글루탐산과 α-케토이소발레르산으로부터 D-발린을 합성하는 내열성 효소를 보유한 고온성 균주 바실러스 LK-2(기탁번호 : KCTC 8717P).Thermophilic strain Bacillus LK-2 with a heat resistant enzyme that synthesizes D-valine from D-glutamic acid and α-ketoisovaleric acid (Accession No .: KCTC 8717P).
KR1019960000709A 1996-01-16 1996-01-16 Bacillus sp. lk-2 having d-alanine aminotransferase KR0184716B1 (en)

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