激光生物学报
激光生物學報
격광생물학보
ACTA LASER BIOLOGY SINICA
2014年
3期
227-233,226
,共8页
近平滑假丝酵母%羰基还原酶%(R)-苯基乙二醇%辅酶再生%酶耦合反应
近平滑假絲酵母%羰基還原酶%(R)-苯基乙二醇%輔酶再生%酶耦閤反應
근평활가사효모%탄기환원매%(R)-분기을이순%보매재생%매우합반응
Candida parapsilosis%carbonyl reductase%(R )-phenyl glycol%cofactor regeneration%enzymatic coupled reaction
经5轮诱变筛选，从近平滑假丝酵母（Candida parapsilosis CICC1676）中分离得到产NADH依赖型羰基还原酶（Carbonyl reductase，CR）菌株CP-9。所产羰基还原酶（CRCp-9）经两步快速纯化获得纯化倍数为11．5倍，比活力为1．84 U/mg的酶液，其还原反应的最适pH值为6．5，最适温度为40℃。该酶转化β-羟基苯乙酮制备手性化合物（R）-苯基乙二醇，因此是（R）-专一性羰基还原酶。该酶与NADH普适性再生酶-甲酸脱氢酶（For-mate dehydrogenase，FDH）在胞外相耦联，构建伴有辅酶再生与反复利用的CR/FDH双酶催化制备立体醇体系，底物β-羟基苯乙酮转化率达95．4％，产物（R）-苯基乙二醇得率为93％，辅酶的总转化数（Total turn number， TTN）达267，产物e．e．值为98．6％，批次耦合反应生产能力达0．8 g/L/h，较单酶催化有较大提高，与细胞转化法相比也具有较好的生产能力。因此，伴有辅酶再生的胞外酶耦合催化具有潜在的制备手性醇化合物的工业应用价值。
經5輪誘變篩選，從近平滑假絲酵母（Candida parapsilosis CICC1676）中分離得到產NADH依賴型羰基還原酶（Carbonyl reductase，CR）菌株CP-9。所產羰基還原酶（CRCp-9）經兩步快速純化穫得純化倍數為11．5倍，比活力為1．84 U/mg的酶液，其還原反應的最適pH值為6．5，最適溫度為40℃。該酶轉化β-羥基苯乙酮製備手性化閤物（R）-苯基乙二醇，因此是（R）-專一性羰基還原酶。該酶與NADH普適性再生酶-甲痠脫氫酶（For-mate dehydrogenase，FDH）在胞外相耦聯，構建伴有輔酶再生與反複利用的CR/FDH雙酶催化製備立體醇體繫，底物β-羥基苯乙酮轉化率達95．4％，產物（R）-苯基乙二醇得率為93％，輔酶的總轉化數（Total turn number， TTN）達267，產物e．e．值為98．6％，批次耦閤反應生產能力達0．8 g/L/h，較單酶催化有較大提高，與細胞轉化法相比也具有較好的生產能力。因此，伴有輔酶再生的胞外酶耦閤催化具有潛在的製備手性醇化閤物的工業應用價值。
경5륜유변사선，종근평활가사효모（Candida parapsilosis CICC1676）중분리득도산NADH의뢰형탄기환원매（Carbonyl reductase，CR）균주CP-9。소산탄기환원매（CRCp-9）경량보쾌속순화획득순화배수위11．5배，비활력위1．84 U/mg적매액，기환원반응적최괄pH치위6．5，최괄온도위40℃。해매전화β-간기분을동제비수성화합물（R）-분기을이순，인차시（R）-전일성탄기환원매。해매여NADH보괄성재생매-갑산탈경매（For-mate dehydrogenase，FDH）재포외상우련，구건반유보매재생여반복이용적CR/FDH쌍매최화제비입체순체계，저물β-간기분을동전화솔체95．4％，산물（R）-분기을이순득솔위93％，보매적총전화수（Total turn number， TTN）체267，산물e．e．치위98．6％，비차우합반응생산능력체0．8 g/L/h，교단매최화유교대제고，여세포전화법상비야구유교호적생산능력。인차，반유보매재생적포외매우합최화구유잠재적제비수성순화합물적공업응용개치。
A bacterial strain CP-9 that produce NADH dependent carbonyl reductase (CR)was isolated from Candida parapsilosis CICC 1676 after 5 cycles mutation and filtration.The produced enzyme CRCp-9 was quickly purified with a 2-step protocol.The protein was purified 1 1 .5-fold and had a specific activity of 1 .84 U/mg.Its optimum reaction pH and temperature of its reduction reaction were identified as 6.5 and 40 ℃ respectively.The CRCp-9 was identified as (R)-specific carbonyl reductase for the chirality compound of (R)-phenyl glycol was produced from the β-hydroxyacetophe-none by the enzyme catalysis.The CRCp-9 was coupled with the universality NADH regenerated enzyme,formate dehydro-genase (FDH)in vitro,thus,CR/FDH system with cofactor regeneration and recycling was constructed to produce ste-reospecific alcohol.The conversion ratio of substrate β-hydroxyacetophenone was 95 .4%,the yield of (R)-phenyl gly-col was 93% with 98.6% e.e.value,the total turn number (TTN)of cofactor was 267,the productivity of one batch coupled reaction reached 0.8 g/L/h.This showed higher productivity than that of single enzymatic catalysis and also had preferable productivity than that of cell conversion.Therefore,producing chirality alcohol compound by the in vitro enzy-matic coupling catalytic with in situ cofactor regeneration has potential application value in industry production.
