乳酸脱氢酶与葡萄糖脱氢酶偶联催化合成D-苯基乳酸
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摘要
D-苯基乳酸(D-phenyllactic acid,D-PLH)是一种理想的天然防腐剂,具有广谱抗菌活性。实验构建了1株乳酸脱氢酶重组大肠杆菌E.coli BL21(DE3)/pET28a-ldh,并对其诱导表达条件进行了优化。在最优条件下(当OD_(600)值达到0.8时,添加乳糖8 g/L,在28℃下诱导8 h),重组乳酸脱氢酶比酶活达到298.8 U/g(干菌体)。建立了乳酸脱氢酶和葡萄糖脱氢酶双酶偶联催化体系,并应用于苯丙酮酸钠还原生成D-苯基乳酸。对反应条件,包括温度、pH值、两重组菌质量比和葡萄糖浓度进行了优化,在最优反应条件下,D-苯基乳酸累积质量浓度达到18.03 g/L,时空产率为162.27 g/(L·d),ee>99%。双酶耦联催化体系是D-苯基乳酸合成的有效工具,显示了良好的应用前景。
D-phenyllactic acid is an ideal natural antimicrobial compound with broad spectrum activity. A lactate dehydrogenase recombinant bacteria Escherichia coli BL21(DE3)/pET28 a-ldh was constructed, and its activity under optimized conditions reached 298.8 U/g dry cell weight. A lactate dehydrogenase and glucose dehydrogenase coupled catalysis system was established and applied in reducing sodium pyruvate to produce D-phenyllactic acid. The reaction conditions were optimized, including temperature, pH, mass ratio of two recombinant bacteria, and glucose concentration. Under the optimal reaction condition, D-phenyllactic acid accumulated up to 18.03 g/L with ee >99%, and the space-time yield reached 162.27 g/(L·d). Therefore, this catalytic system is effective to synthesize D-phenyllactic acid and has shown a good application prospect.
D-phenyllactic acid is an ideal natural antimicrobial compound with broad spectrum activity. A lactate dehydrogenase recombinant bacteria Escherichia coli BL21(DE3)/pET28 a-ldh was constructed, and its activity under optimized conditions reached 298.8 U/g dry cell weight. A lactate dehydrogenase and glucose dehydrogenase coupled catalysis system was established and applied in reducing sodium pyruvate to produce D-phenyllactic acid. The reaction conditions were optimized, including temperature, pH, mass ratio of two recombinant bacteria, and glucose concentration. Under the optimal reaction condition, D-phenyllactic acid accumulated up to 18.03 g/L with ee >99%, and the space-time yield reached 162.27 g/(L·d). Therefore, this catalytic system is effective to synthesize D-phenyllactic acid and has shown a good application prospect.
引文
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[23] LIU W F,WANG P.Cofactor regeneration for sustainable enzymatic biosynthesis[J].Biotechnology Advances,2007,25:369-384.
[24] TANG Y P,ZHANG G M,WANG Z,et al.Efficient synthesis of a (S)-fluoxetine intermediate using carbonyl reductase coupled with glucose dehydrogenase[J].Bioresource Technology,2018,250:457-463.
[25] 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,2017,33:1 235-1 242.
[26] LIU J,PANG BQ,ADAMS J P,et al.Coupled immobilized amine dehydrogenase and glucose dehydrogenase for asymmetric synthesis of amines by reductive amination with cofactor recycling[J].Chemcatchem,2017,9:425-431.
[2] MU W M,YU S H,ZHU L J,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.
[3] LI R,SUN X,XU Y,et al.Novel antimicrobial and antioxidant chitosan derivatives prepared by green grafting with phenyllactic acid[J].Food Biophysics,2017,12(4):470-478.
[4] WANG M,ZHU L F,XU X L,et al.Efficient production of enantiomerically pure D-phenyllactate from phenylpyruvate by structure-guided design of an engineered D-lactate dehydrogenase[J].Applied Microbiology and Biotechnology,2016,100(17):7 471-7 478.
[5] 胡发根.重组大肠杆菌全细胞转化合成D-苯基乳酸的基础研究[D].苏州:苏州大学,2015.
[6] WANG J P,YOO J S,LEE J H,et al.Effects of phenyllactic acid on growth performance,nutrient digestibility,microbial shedding,and blood profile in pigs[J].Journal of Animal Science,2009,87(10):3 235-3 243.
[7] WANG J P,Yoo J S,LEE J H,et al.Effects of phenyllactic acid on production performance,egg quality parameters,and blood characteristics in laying hens[J].The Journal of Applied Poultry Research,2009,18(2):203-209.
[8] KAWAGUCHI H,TERAMURA H,UEMATSU K,et al.Phenyllactic acid production by simultaneous saccharification and fermentation of pretreated sorghum bagasse[J].Bioresource Technology,2015,182:169-178.
[9] FUJITA T,HIEU D N,ITO T,et al.Microbial monomers custom-synthesized to build true bio-derived aromatic polymers[J].Applied Microbiology and Biotechnology,2013,97:8 887-8 894.
[10] ZHENG Z J,SHENG B B,GAO C,et al.Highly stereoselective biosynthesis of (R)-alpha-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase[J].Scientific Reports,2013,3:3 401.
[11] 李德茂,李从发,刘四新,等.3-苯基乳酸的研究进展[J].药物生物技术,2004,11(5):344-347.
[12] KANO S,YUASA Y,YOKOMATSU T,et al.Highly stereocontrolled synthesis of the four individual stereoisomers of statine[J].The Journal of Organic Chemistry,1998,53(16):3 865-3 868.
[13] HASHIMOTO Y,KOBAYASHI E,ENDO T,et al.Conversion of a cyanhydrin compound into S-3-phenyllactic acid by enantioselective hydrolytic activity of Pseudomonas sp.BC-18[J].Bioscience,Biotechnology,and Biochemistry,1996,60(8):1 279-1 283.
[14] XU G C,ZHANG L L,NI Y.Enzymatic preparation of D-phenyllactic acid at high space-time yield with a novel phenylpyruvate reductase identified from Lactobacillus sp.CGMCC 9967[J].Journal of Biotechnology,2016,222:29-37.
[15] ZHU Y B,HU F G,ZHU Y Y,et al.Enhancement of phenyllactic acid biosynthesis by recognition site replacement of D-lactate dehydrogenase from Lactobacillus pentosus[J].Biotechnology Letters,2015,37(6):1 233-1 241.
[16] ZHOU X H,ZHOU J,XIN F X,et al.Heterologous expression of a novel d-lactate dehydrogenase from Lactobacillus sp.ZX1 and its application for D-phenyllactic acid production[J].International Journal of Biological Macromolecules,2018,119:1 171-1 178.
[17] FUJII T,SHIMIZU M,DOI Y,et al.Novel fungal phenylpyruvate reductase belongs to d-isomer-specific 2-hydroxyacid dehydrogenase family[J].Biochimica Et Biophysica Acta-Proteins and Proteomics,2011,1814(12):1 669-1 676.
[18] ANA B Y,GRACIELA F,CARLA L G.Optimization of phenyllactic acid production by Pediococcus acidilactici CRL 1753.Application of the formulated bio-preserver culture in bread[J].Biological Control,2018,123:137-143.
[19] YU S H,ZHU L J,ZHOU C,et al.Enzymatic production of D-3-phenyllactic acid by Pediococcus pentosaceus D-lactate dehydrogenase with NADH regeneration by Ogataea parapolymorpha formate dehydrogenase[J].Biotechnology Letters,2014,36(3):627-631.
[20] ZHU Y B,XU Y,WANG L M,et al.Biosynthesis of (R)-2-hydroxy-3-phenylpropionic acid using whole recombinant Escherichia coli cells in an aqueous/n-octane biphasic system[J].Journal of Zhejiang University-Science B,2018,19:285-292.
[21] NANBA H,TAKAOKA Y,HASEGAWA J.Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M,and cloning of the gene[J].Bioscience Biotechnology and Biochemistry,2003,67(4):720-728.
[22] WICHMANN R,VASIC-RACKI D.Cofactor regeneration at the lab scale[J].Advances in Biochemical Engineering-Biotechnology,2005,92:225-260.
[23] LIU W F,WANG P.Cofactor regeneration for sustainable enzymatic biosynthesis[J].Biotechnology Advances,2007,25:369-384.
[24] TANG Y P,ZHANG G M,WANG Z,et al.Efficient synthesis of a (S)-fluoxetine intermediate using carbonyl reductase coupled with glucose dehydrogenase[J].Bioresource Technology,2018,250:457-463.
[25] 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,2017,33:1 235-1 242.
[26] LIU J,PANG BQ,ADAMS J P,et al.Coupled immobilized amine dehydrogenase and glucose dehydrogenase for asymmetric synthesis of amines by reductive amination with cofactor recycling[J].Chemcatchem,2017,9:425-431.
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