分枝杆菌LY-1转化植物甾醇产9α羟基雄烯二酮的发酵工艺优化
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摘要
9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)是一种重要的甾体药物中间体,可用于糖皮质激素类药物的生产。为了提高目的产物的产量,以分枝杆菌(Mycobacterium sp.) LY-1作为出发菌株,研究不同碳源、氮源、磷酸盐和金属离子等培养基组分对菌株LY-1转化植物甾醇生产9α-OH-AD的影响,结合正交试验优化,确定最适培养基组分为玉米浆30 g/L、KNO_3 4.0 g/L、NaH_2PO_4 1.2 g/L、FeSO_4 0.075 g/L及CaCl_2 0.10 g/L。在此条件下,当植物甾醇投料质量浓度为15 g/L时,产物得率达到43.9%～44.9%。通过单因素实验优化培养条件,得到最适培养条件为pH 8.0、接种量2%、温度30℃时,9α-OH-AD的得率可达46.2%～47.0%,比优化前提高了22.1%,产量达到5.21 g/L,具有潜在的工业应用价值。
9α-Hydroxy-4-androstene-3,17-dione(9α-OH-AD) is an important precursor to produce glucocorticoid drugs. To improve the yield of 9α-OH-AD,Mycobacterium sp. LY-1 was used as the wild strain. To study the effects of different carbon sources,nitrogen source,phosphate and inorganic salts on the concentration of 9α-OH-AD, we optimized the medium by orthogonal experiments.The optimized medium was composed as(g/L):corn steep liquor 30,KNO_3 4.0,NaH_2PO_4 1.2,FeSO_4 0.075,and CaCl_2 0.10. Under this condition,the yield of 9α-OH-AD reached 43.9%-44.9% when the concentration of phytosterol was 15 g/L. Then,the conditions of culture were optimized through single factors experiments. The suitable culture conditions were determined as:pH 8.0,2% inoculum concentration and 30 ℃. Under these conditions,the yield of 9α-OH-AD reached 46.2%-47.0%,increased by 22.1% compared with the control,and the concentration of 9α-OH-AD was 5.21 g/L. It has a potential industrial application.
9α-Hydroxy-4-androstene-3,17-dione(9α-OH-AD) is an important precursor to produce glucocorticoid drugs. To improve the yield of 9α-OH-AD,Mycobacterium sp. LY-1 was used as the wild strain. To study the effects of different carbon sources,nitrogen source,phosphate and inorganic salts on the concentration of 9α-OH-AD, we optimized the medium by orthogonal experiments.The optimized medium was composed as(g/L):corn steep liquor 30,KNO_3 4.0,NaH_2PO_4 1.2,FeSO_4 0.075,and CaCl_2 0.10. Under this condition,the yield of 9α-OH-AD reached 43.9%-44.9% when the concentration of phytosterol was 15 g/L. Then,the conditions of culture were optimized through single factors experiments. The suitable culture conditions were determined as:pH 8.0,2% inoculum concentration and 30 ℃. Under these conditions,the yield of 9α-OH-AD reached 46.2%-47.0%,increased by 22.1% compared with the control,and the concentration of 9α-OH-AD was 5.21 g/L. It has a potential industrial application.
引文
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[20] EGOROVA O V,NIKOLAVEVA V M,SUKHODOLSKAVA G V,et al.Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp.[J].J Mol Catal B:Enzymatic,2009,57:198-203.
[21] 薛凯,王荣,王友富,等.响应面法优化植物甾醇转化制备9-羟基-雄烯二酮[J].食品工业,2017(9):169-173.
[22] GUEVARA G,HERAS L F L,PERERA J,et al.Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcusruber strain Chol-4[J].J Steroid Biochem Mol Biol,2017,172:176-187.
[23] PETRUSMA M,GEIZE R V D,DIJKHUIZEN L.3-Ketosteroid 9α-hydroxylase enzymes:rieske non-heme monooxygenases essential for bacterial steroid degradation[J].Antonie Van Leeuwenhoek,2014,106(1):157-72.
[24] 袁家代,陈贵英,程世君,等.3-甾酮-9α-羟基化酶基因在分枝杆菌中的异源表达与9α-羟基雄烯二酮的制备[J].生物工程学报,2015,31(4):523-533.
[25] 王贵娥,陈艺强,范永仙,等.3-甾酮-9α-羟基化酶基因在分枝杆菌中强化表达[J].药物生物技术,2016(5):381-384.
[2] LEDNICER D.Steroid chemistry at a glance[M].New York:John Wiley & Sons,Ltd.,2011.
[3] 张裕卿,王东青.植物甾醇微生物转化制备甾体药物中间体的研究进展[J].微生物学通报,2006,33(2):142-146.
[4] MORIARTY R M,RAO P.Methods for preparing synthetic bile acids and compositions comprising the same:WO,2012047495[P].2012-04-12.
[5] TONG W Y,DONG X.Microbial biotransformation:recent developments on steroid drugs[J].Recent Patents Biotechnol,2009,3(2):141-153.
[6] WEI W,FAN S Y,WANG F Q,et al.A new steroid-transforming strain of Mycobacterium neoaurum and cloning of 3-ketosteroid 9α-hydroxylase in NwIB-01[J].Appl Biochem Biotechnol,2010,162(5):1446-1456.
[7] GAO X Q.Enhanced steroid metabolites production by resting cell phytosterol bioconversion[J].Chem Biochem Eng Q,2016,29 (4):567-573.
[8] YUAN J J,GUAN Y X,WANG Y T,et al.Side-chain cleavage of phytosterols by Mycobacterium sp.MB 3683 in a biphasic ionic liquid/aqueous system[J].J Chem Technol Biotechnol,2016,91(10):2631-2637.
[9] DONOVA M V,GULEVSKAYA S A,DOVBNYA D V.Mycobacterium sp.mutant strain producing 9α-hydroxy-androstenedione from sitosterol[J].Appl Microbiol Biotechnol,2005,67:671-678.
[10] 徐阳光.分枝杆菌降解植物甾醇侧链过程基础研究[D].杭州:浙江大学,2014.
[11] 柳相鹤,张瑞婕,赵树欣,等.Mycobacterium sp.BFZ304转化植物甾醇产9α-羟基雄烯二酮培养基的响应面优化[J].食品工业科技,2016,37(16):172-177.
[12] 罗炳华.外加因子对两相系统转化植物甾醇制备雄烯二酮的影响[D].合肥:合肥工业大学,2009.
[13] 王艳婷,袁俊杰,关怡新,等.双水相体系中分枝杆菌降解植物甾醇制备雄甾-4-烯-3,17-二酮[J].高校化学工程学报,2017,31(1):104-110.
[14] 张磊.微生物转化法生产雄烯二酮工艺条件优化[D].郑州:河南大学,2016.
[15] 宋宇迪,董玉秀,马新宇,等.降解植物甾醇为甾体9α-OH-AD菌株的筛选与鉴定[J].应用与环境生物学报,2017(6):1022-1027.
[16] PATTANA S,AURASORN S.Utilization of phytosterol-containing vegetable oils as a substrate for production of androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione by using Mycobacterium sp.[J].Biocatal Agric Biotechnol,2016,8:18-23.
[17] YAO K,XU L Q,WANG F Q,et al.Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols[J].Metab Eng,2014,24:181-191.
[18] 诸葛健,李华钟.微生物学[M].2版.北京:科学出版社,2009.
[19] 姜博,谢定,郑瑞娜,等.海藻糖合成酶基因工程菌玉米浆培养基配方优化[J].食品与机械,2016,32(11):183-186.
[20] EGOROVA O V,NIKOLAVEVA V M,SUKHODOLSKAVA G V,et al.Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp.[J].J Mol Catal B:Enzymatic,2009,57:198-203.
[21] 薛凯,王荣,王友富,等.响应面法优化植物甾醇转化制备9-羟基-雄烯二酮[J].食品工业,2017(9):169-173.
[22] GUEVARA G,HERAS L F L,PERERA J,et al.Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcusruber strain Chol-4[J].J Steroid Biochem Mol Biol,2017,172:176-187.
[23] PETRUSMA M,GEIZE R V D,DIJKHUIZEN L.3-Ketosteroid 9α-hydroxylase enzymes:rieske non-heme monooxygenases essential for bacterial steroid degradation[J].Antonie Van Leeuwenhoek,2014,106(1):157-72.
[24] 袁家代,陈贵英,程世君,等.3-甾酮-9α-羟基化酶基因在分枝杆菌中的异源表达与9α-羟基雄烯二酮的制备[J].生物工程学报,2015,31(4):523-533.
[25] 王贵娥,陈艺强,范永仙,等.3-甾酮-9α-羟基化酶基因在分枝杆菌中强化表达[J].药物生物技术,2016(5):381-384.
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