Lactobacillus plantarum苯丙酮酸还原酶的异源表达及其在苯乳酸制备中的应用
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摘要
构建产苯丙酮酸还原酶的基因工程菌E.coli/ppr,优化其目的蛋白表达条件,并利用全细胞催化制备光学纯的D-苯乳酸。以Lactobacillus plantarum的基因组DNA为模板,经PCR扩增出一种编码苯丙酮酸还原酶的基因(ppr),并将其在大肠杆菌BL21中进行表达。以苯丙酮酸为底物,通过单因素和正交试验优化诱导表达条件,然后对E.coli/ppr全细胞制备苯乳酸的工艺进行研究。结果表明:E.coli/ppr在IPTG终浓度为0.5 mmol/L,20℃诱导8 h时具有最高的苯丙酮酸还原酶活性。由其催化制备苯乳酸的最佳条件为:反应温度40℃,反应初始pH 6.5,葡萄糖浓度20 mmol/L。在上述条件下增加苯丙酮酸的浓度至25 mmol/L,5 h后苯乳酸的最终产率达到98.4%,产物D-苯乳酸光学纯度ee>99.9%。
In order to construct a genetic engineering phenylpyruvate reductase strain( E.coli/ppr),the expression conditions and bioconversion medium were optimized to increase the production of PLA with whole-cell transformation.A phenylpyruvate reductase-encoding gene,ppr,was obtained by PCR from the genomic DNA of Lactobacillus plantarum and heterologously expressed in E.coli BL21( DE3).Taking phenylpyruvic acid as the substrate,single factor and orthogonal experiments was used to optimized the induced expression and whole-cell catalysis conditions.Results showed that after induction with 0.5 mmol/L IPTG at 20 ℃ for 8 h,E.coli/ppr displayed the highest Lp PPR activity.When phenylpyruvic acid was added up to 25 mmol/L,the product enantiomeric excess percent was over 99.9% and the final yield of PLA could reach 98.38% at 5 h under the optimal catalytic conditions as follows: temperature 40 ℃,initial pH 6.5 and 20 mmol/L glucose.
In order to construct a genetic engineering phenylpyruvate reductase strain( E.coli/ppr),the expression conditions and bioconversion medium were optimized to increase the production of PLA with whole-cell transformation.A phenylpyruvate reductase-encoding gene,ppr,was obtained by PCR from the genomic DNA of Lactobacillus plantarum and heterologously expressed in E.coli BL21( DE3).Taking phenylpyruvic acid as the substrate,single factor and orthogonal experiments was used to optimized the induced expression and whole-cell catalysis conditions.Results showed that after induction with 0.5 mmol/L IPTG at 20 ℃ for 8 h,E.coli/ppr displayed the highest Lp PPR activity.When phenylpyruvic acid was added up to 25 mmol/L,the product enantiomeric excess percent was over 99.9% and the final yield of PLA could reach 98.38% at 5 h under the optimal catalytic conditions as follows: temperature 40 ℃,initial pH 6.5 and 20 mmol/L glucose.
引文
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[3]LAVERMICOCCA P,VALERIO F,VISCONTI A.Antifungal activity of phenyllactic acid against molds isolated from bakery products[J].Applied and Environmental Microbiology,2003,69(1):634-640.
[4]FUJITA T,NGUYEN H D,ITO T,et al.Microbial mono-mers custom-synthesized to build true bio-derived aromatic polymers[J].Applied Microbiology and Biotechnology,2013,97(20):8 887-8 894.
[5]MU Wan-meng,LIU Feng-li,JIA Jiang-hua,et al.3-Phenyllactic acid production by substrate feeding and p H-control in fed-batch fermentation of Lactobacillus sp.SK007[J].Bioresource Technology,2009,100(21):5 226-5 229.
[6]MU Wan-meng,CHEN Chao,LI Xing-Feng,et al.Optimization of culture medium for the production of phenyllactic acid by Lactobacillus sp.SK007[J].Bioresource Technology,2009,100(3):1 366-1 370.
[7]RODRIGUEZ-PAZO N,VAZQUEZ-ARAUJO L,PEREZRODRIGUEZ N,et al.Cell-free supernatants obtained from fermentation of cheese whey hydrolyzates and phenylpyruvic acid by Lactobacillus plantarum as a source of antimicrobial compounds,bacteriocins,and natural aromas[J].Applied Biochemistry and Biotechnology,2013,171(4):1 042-1 060.
[8]MU Wan-meng,YU Shu-huai,JIANG Bo,et al.Characterization of D-lactate dehydrogenase from Pediococcus acidilactici that converts phenylpyruvic acid into phenyllactic acid[J].Biotechnology Letters,2012,34(5):907-911.
[9]王颖,范铭,薛素妹,等.全细胞催化合成L-苯基乳酸重组大肠杆菌的构建[J].食品与发酵工业,2015,41(12):13-17.
[10]张玲玲,许国超,倪晔.不对称合成苯乳酸的酮酸还原酶基因克隆和表达[J].食品与生物技术学报,2016,35(9):950-957.
[11]MU Wan-meng,YU Shu-huai,ZHU Lan-jun,et al.Recent research on 3-phenyllactic acid,a broad-spectrum antimicrobial compound[J].Applied Microbiology and Biotechnology,2012,95(5):1 155-1 163.
[12]MALAKAR P,VENKATESH K V.Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins[J].Applied Microbiology and Biotechnology,2012,93(6):2 543-2 549.
[13]BALZER G J,THAKKER C,BENNETT G N,et al.Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase[J].Metabolic Engineering,2013,20:1-8.
[14]ZHENG Zhao-juan,MA Cui-qing,GAO Chao,et al.Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM[J].PLo S One,2011,6(4):e19030.
[15]LI L,SHIN S-Y,LEE K W,et al.Production of natural antimicrobial compound D-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active D-lactate dehydrogenase[J].Letters in Applied Microbiology,2014,59(4):404-411.