裂殖酵母醛酮还原酶基因的克隆与诱导表达条件优化
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摘要
从裂殖酵母(Schizosaccharomyces pombe)基因组中克隆获得编码醛酮还原酶SpoAKR3C2的基因,并成功构建了表达SpoAKR3C2的重组工程菌Escherichia coli BL21 (DE3)/pET 28bSpoAKR3C2.为了提高SpoAKR3C2在E.coli BL21(DE3)中的表达水平,分别考察了种子液接种量、诱导时间、温度、转速以及诱导剂浓度等对产醛酮还原酶SpoAKR3C2的影响.结果表明:该酶的最适诱导温度为19℃,最适转速为140r/min,最适诱导时间为14h,最适接种量为1.5%(体积比).以IPTG为诱导剂,最适诱导剂浓度为0.1 mmol/L.在最佳诱导条件下,粗酶液中SpoAKR3C2对酮基泛解酸内酯的比酶活可达6.762U/mg,与未优化前相比提高了3.4倍.
The gene encoding an aldo-keto reductase(SpoAKR3 C2)from Schizosaccharomyces pombe was cloned and over-expressed in Escherichia coli BL21(DE3).The effects of inoculation amount,time,temperature,rotation speed and inducer concentration on the expression of SpoAKR3 C2 were studied.The results showed that the optimal induction temperature was 19℃,the optimal rotation speed was 140 r/min,the optimum induction time was 14 h,and the optimum inoculation amount was 1.5%(volume ratio).The optimum IPTG concentration was 0.1 mmol/L.Under optimized induction conditions,the SpoAKR3 C2 activity in crude cell extracts was 6.762 U/mg using ketopantolactone as substrate,which was 3.4 times higher than that before the optimization.
The gene encoding an aldo-keto reductase(SpoAKR3 C2)from Schizosaccharomyces pombe was cloned and over-expressed in Escherichia coli BL21(DE3).The effects of inoculation amount,time,temperature,rotation speed and inducer concentration on the expression of SpoAKR3 C2 were studied.The results showed that the optimal induction temperature was 19℃,the optimal rotation speed was 140 r/min,the optimum induction time was 14 h,and the optimum inoculation amount was 1.5%(volume ratio).The optimum IPTG concentration was 0.1 mmol/L.Under optimized induction conditions,the SpoAKR3 C2 activity in crude cell extracts was 6.762 U/mg using ketopantolactone as substrate,which was 3.4 times higher than that before the optimization.
引文
[1]史丽珍,应彬彬,王亚军.微生物醛酮还原酶结构、功能及其在生物催化中的应用[J].发酵科技通讯,2017,46(4):198-204.
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[6]应向贤,高亮,张丽,等.酿酒酵母醛酮还原酶的克隆表达和酶学性质研究[J].发酵科技通讯,2017,46(3):129-133.
[7]廖锡豪,陈明祥,谢万勇,等.低温诱导对毕赤酵母表达重组外源蛋白的影响[J].中国酿造,2013,32(2):9-12.
[8] PATNAIK P R.Investigation of induction effect on the steady state performance of a continuous fermentation for recombinantβ-ga1actosidase[J].Process biochemistry,2001,36(11):1069-1074.
[9]张金红,陈华友,李萍萍.基因工程菌发酵研究进展[J].生物学杂志,2012,19(5):72-75.
[2] WANG Y J,SHEN W,LUO X,et al.Enhanced diastereoselective synthesis of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate by using aldo-keto reductase and glucose dehydrogenase co-producing engineered Escherichia coli[J].Biotechnology progress,2017,33(5):1235-1242.
[3]汪钊,黄美娟,高亮,等.化学酶法合成D-泛解酸内酯的研究进展[J].发酵科技通讯,2016,45(4):193-198.
[4] SI D,URANO N,NOZAKI,et al.L-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis:genetic analyses and application to the stereospecific oxidation of L-pantoyl lactone[J].Applied microbiology and biotechnology,2012,95(2):431-440.
[5] ZHAO M,GAO L,ZHANG L,et al.Efficient asymmetric reduction of ketopantolactone using a strictly(R)-stereoselective carbonyl reductase through the substrate constant-feeding strategy[J].Biotechnology letter,2017,39(11):1741-1746.
[6]应向贤,高亮,张丽,等.酿酒酵母醛酮还原酶的克隆表达和酶学性质研究[J].发酵科技通讯,2017,46(3):129-133.
[7]廖锡豪,陈明祥,谢万勇,等.低温诱导对毕赤酵母表达重组外源蛋白的影响[J].中国酿造,2013,32(2):9-12.
[8] PATNAIK P R.Investigation of induction effect on the steady state performance of a continuous fermentation for recombinantβ-ga1actosidase[J].Process biochemistry,2001,36(11):1069-1074.
[9]张金红,陈华友,李萍萍.基因工程菌发酵研究进展[J].生物学杂志,2012,19(5):72-75.