为了提高来源于地衣芽孢杆菌(Bacillus licheniformis)的乙酰乳酸合成酶(acetolactate synthetase, ALS)的产酶水平,将含有目的基因的重组质粒pEGX-6p-1-als S,转化到E.coli BL21 (DE3)进行异源表达,并对重组alsS的发酵条件进行优化。发酵结果显示,优化的发酵培养基成分包括葡萄糖12 g/L、蛋白胨10 g/L、柠檬酸钠5 g/L、KH2PO4 3 g/L、MgSO4·7H2O 1 g/L和NaCl 2 g/L;在优化的培养基中,最佳诱导条件为诱导温度 30 ℃,异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)浓度 0.4 mmol/L,诱导时间10 h。通过响应面预测发现,最佳产酶水平条件为pH 7.0、温度30.2 ℃,IPTG浓度为0.42 mmol/L。经发酵试验验证,该条件下产酶水平达到242.567 U/mL。基于以上所有优化条件,单位菌体(干重)产乙酰乳酸合成酶量达164.299 kU/g,产酶水平达266.657 U/mL,分别是LB培养基的1.31和 1.77倍。
This paper aimed to improve the enzyme production level of acetolactate synthetase (ALS) from Bacillus licheniformis. The recombinant plasmid pEGX-6p-1-alsS was transformed into Escherichia coli BL21 (DE3) for heterologous expression, and the fermentation conditions of recombinant E. coli BL21 (DE3) were optimized. The optimized fermentation medium was composed of glucose 12 g/L, peptone 10 g/L, sodium citrate 5 g/L, KH2PO4 3 g/L, MgSO4·7H2O 1 g/L and NaCl 2 g/L. Moreover, induction with 0.4 mmol/L isopropyl-β-D-thiogalactopyranoside at 30 ℃ for 10 h was considered as the optimized condition. Based on the response surface prediction, the optimal conditions for enzyme production were pH 7.0, temperature 30.2 ℃, and 0.42 mmol/L IPTG. Under this condition, the enzyme production level was 242.567 U/mL. Therefore, the amount of ALS produced by unit bacteria (dry weight) was 164.299 kU/g, and the enzyme production level was 266.657 U/mL, which were 1.31 and 1.77 times of that of the lysogeny broth (LB) medium, respectively.
[1] 崔真真,毛雨丰,陈聪,等.生物法合成双乙酰的研究进展[J].生物加工过程,2017,15(5):57-64.
[2] BENSON K H, GODON J J, RENAULT P, et al. Effect of ilvBN-encoded α-acetolactate synthase expression on diacetyl production in Lactococcus lactis[J]. Applied Microbiology and Biotechnology, 1996, 45(1-2): 107-111.
[3] HUGENHOLTZ J, KLEEREBEZEM M, STARRENBURG M, et al. Lactococcus lactis as a cell factory for high-level diacetyl production [J]. Applied Microbiology and Biotechnology, 2000, 66(9): 4 112-4 114.
[4] MAN J C D. The formation of diacetyl and acetoin from α-acetolactic acid [J]. 1959, 78(7):480-486.
[5] 赵宏峰.乳酸乳球菌α-乙酰乳酸合成酶基因的克隆及在大肠杆菌中的表达[D], 上海:复旦大学, 2010.
[6] GUPTA K G, YADAVN K, DHAWAN S. Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613[J]. Biotechnology and Bioengineering, 1978, 20(12): 1 895-1 901.
[7] JYOTI B D, SURESH A K, VENKATESH K V. Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates [J]. World Journal of Microbiology & Biotechnology, 2003, 19(5): 509-514.
[8] CHUANG L F, COLLINS E B. Biosynthesis of diacetyl in bacteria and yeast[J]. 1968, 95(6): 2 083-2 089.
[9] 黄彦,韦宇拓,张黎,等. 乳酸片球菌L-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达[J]. 应用与环境生物学报,2006,12(1): 68-71.
[10] ZHAO L, BAO Y, WANG J, et al. Optimization and mechanism of diacetyl accumulation by Enterobacter aerogenes mutant UV-3[J]. World Journal of Microbiology & Biotechnology, 2009, 25(1): 57-64.
[11] GAO X, XU N, LI S, et al. Metabolic engineering of Candida glabrata for diacetyl production[J]. PLOS ONE,2014,9(3): E89 854.
[12] SINGH B K, STIDHAM M A, SHANER D L. Assay of acetohydroxyacid synthase[J]. Analytical biochemistry, 1988, 171(1): 173-179.
[13] 金美娟,吴坚平,徐刚,等. 乙酰乳酸合成酶基因的克隆与高效表达[J]. 微生物学通报,2012,39(11): 1 589-1 596.
[14] 史永磊. 南极微生物SOD基因克隆与表达及发酵优化的研究[D]. 哈尔滨:哈尔滨工业大学, 2015.
[15] 边雅倩,许国超,倪晔. 重组大肠杆菌产双乙酰还原酶的发酵条件优化[J]. 食品与生物技术学报,2017,36(9): 54-60.
[16] 钱铭镛. 微生物产酶的控制[J]. 江苏食品与发酵,1982(3):29-31.
[17] 刘颖,张彬彬,孙冰玉,等.枯草芽孢杆菌高产中性蛋白酶发酵条件的优化[J]. 食品科学,2014,35(13):166-170.
[18] 黄辉,汤斌.基于响应面法对枯草芽孢杆菌WB600-eg2发酵产内切葡聚糖苷酶条件的优化[J]. 安徽工程大学学报,2012,27(1):1-4;13.
[19] 王东,荣家萍,唐自钟,等. 响应面法优化枯草芽孢杆菌产中性蛋白酶的发酵条件[J]. 基因组学与应用生物学,2016,35(1):143-151.
