重组大肠杆菌全细胞合成D-苯基乳酸
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摘要
苯基乳酸(phenyllactic acid,PLA)是一种重要的有机酸,广泛应用于食品、医药、化妆品等领域。通过构建D-乳酸脱氢酶重组大肠杆菌BL21(DE3)/p ET28a-ldh D利用全细胞催化合成D-苯基乳酸。对重组大肠杆菌的诱导及转化条件进行了优化,最终确定了最佳的诱导条件:OD600=0. 8时,添加IPTG至终浓度为0. 2 mmol/L,在25℃下诱导4 h;最佳的转化条件:pH 7. 0磷酸缓冲液,13 g/L苯丙酮酸钠,5 g/L葡萄糖,30 g/L菌体(干重),37℃,200 r/min,转化2 h。在最优的条件下,苯基乳酸的产物浓度达到5. 2 g/L,产率为40%。该文还探索了苯丙酮酸钠与葡萄糖分批添加对苯基乳酸合成的影响,并初步得到了最佳工艺,经3 h转化,苯基乳酸产物质量浓度和产率分别提高到7. 38 g/L和52. 7%,显示了较好的应用前景,也为后续酶的改造奠定了基础。
Phenyllactic acid is an important organic acid that has been widely used in foods,pharmaceuticals,cosmetics and other fields. D-phenyllactic acid was catalytically synthesized by whole cells through constructing recombinant Escherichia coli BL21(DE3)/p ET28 a-ldh D. The induction and transformation conditions were optimized and determined as follows: IPTG was added to a final concentration of 0. 2 mmol/L when OD600 reached 0. 8 and cells were induced at 25 ℃ for 4 h. The optimal transformation conditions were as follows: using pH = 7. 0 phosphate buffer,13 g/L sodium phenylpyruvate,5 g/L glucose,30 g/L cells(dry weight),at 37 ℃ and 200 r/min for 2 h. Under the optimal conditions,phenyllactic acid accumulated up to 5. 2 g/L and the yield was 40%. Effects of fed-batch process using sodium pyruvate and glucoase on phenyllactic acid synthesis were explored,and the best processing scheme was obtained. After 3 h of conversion,the concentration of D-phenyllactic and its yield increased to 7. 38 g/L and 52. 7%,respectively. This study showed that the process of whole cell transformation for D-phenyllactic acid synthesis had good application prospects,which laid a foundation for subsequent enzymatic modification.
Phenyllactic acid is an important organic acid that has been widely used in foods,pharmaceuticals,cosmetics and other fields. D-phenyllactic acid was catalytically synthesized by whole cells through constructing recombinant Escherichia coli BL21(DE3)/p ET28 a-ldh D. The induction and transformation conditions were optimized and determined as follows: IPTG was added to a final concentration of 0. 2 mmol/L when OD600 reached 0. 8 and cells were induced at 25 ℃ for 4 h. The optimal transformation conditions were as follows: using pH = 7. 0 phosphate buffer,13 g/L sodium phenylpyruvate,5 g/L glucose,30 g/L cells(dry weight),at 37 ℃ and 200 r/min for 2 h. Under the optimal conditions,phenyllactic acid accumulated up to 5. 2 g/L and the yield was 40%. Effects of fed-batch process using sodium pyruvate and glucoase on phenyllactic acid synthesis were explored,and the best processing scheme was obtained. After 3 h of conversion,the concentration of D-phenyllactic and its yield increased to 7. 38 g/L and 52. 7%,respectively. This study showed that the process of whole cell transformation for D-phenyllactic acid synthesis had good application prospects,which laid a foundation for subsequent enzymatic modification.
引文
[1]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.
[2] DIEULEVEUX V,GUEGUEN M. Antimicrobial effects of D-3-phenyllactic acid on Listeria monocytogenes in TSBYEmedium,milk,and cheese[J]. Journal of Food Protection,1998,61(10):1 281-1 285.
[3] DIEULEVEUX V,LEMARINIER S,GUEGUEN M. Antimicrobialspectrum and target site of D-3-phenyllactic acid[J]. International Journal of Food Microbiology,1998,40(3):177-183.
[4] VALERIO F,LAVERMICOCCA P,PASCALE M. Production of phenyllactic acid by lactic acid bacteria:an approach to the selection of strains contributing to food quality and preservation[J]. FEMS Microbiology Letters,2004,233(2):289-295.
[5] 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.
[6]李兴峰,江波,潘蓓蕾,等.新型生物防腐剂—苯乳酸在食品中的研究与应用[J].食品与发酵工业,2007,33(5):87-91.
[7] WANG J P,YOO J S,LEE J H. 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.
[8]江明华,林华,施勤,等.β-苯基乳酸对离体小鼠子宫的平滑肌作用[J].上海医科大学学报,1990,17(5):350-354.
[9]王珏英,邵以德,张渊博,等.β-苯基乳酸对心血管系统的实验研究[J].上海医科大学学报,1991,18(4):295-297.
[10]程彰华,楼亚平,戴华娟,等.丹参素对微循环障碍和血浆乳酸含量影响的实验研究[J].上海医科大学学报,1987,14(1):25-29.
[11] HASHIMOTO Y,KOBAYASHI E,ENDO T. 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.
[12]孟琳,白云平,葛双启.果酸在化妆品中的应用[J].化学与生物工程,2005,22(4):45-46.
[13]贾江花.乳酸脱氢酶的克隆表达及酶学性质的研究[D].无锡:江南大学,2009:25-26.
[14] RODRIGUEZ N,SALGADO JM,CORTES S. Antimicrobial activity of D-3-phenyllactic acid produced by fedbatch process against Salmonella enterica[J]. Food Control,2012,25(1):274-284.
[15]赵明月,郑兆娟.重组大肠杆菌全细胞催化合成D-苯基乳酸的研究[J].广东化工,2015,42(6):29-30.
[16]黄志华,刘铭,王宝光,等.甲酸脱氢酶用于辅酶NADH再生的研究进展[J].过程工程学报,2006,6(6):1 011-1017.
[2] DIEULEVEUX V,GUEGUEN M. Antimicrobial effects of D-3-phenyllactic acid on Listeria monocytogenes in TSBYEmedium,milk,and cheese[J]. Journal of Food Protection,1998,61(10):1 281-1 285.
[3] DIEULEVEUX V,LEMARINIER S,GUEGUEN M. Antimicrobialspectrum and target site of D-3-phenyllactic acid[J]. International Journal of Food Microbiology,1998,40(3):177-183.
[4] VALERIO F,LAVERMICOCCA P,PASCALE M. Production of phenyllactic acid by lactic acid bacteria:an approach to the selection of strains contributing to food quality and preservation[J]. FEMS Microbiology Letters,2004,233(2):289-295.
[5] 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.
[6]李兴峰,江波,潘蓓蕾,等.新型生物防腐剂—苯乳酸在食品中的研究与应用[J].食品与发酵工业,2007,33(5):87-91.
[7] WANG J P,YOO J S,LEE J H. 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.
[8]江明华,林华,施勤,等.β-苯基乳酸对离体小鼠子宫的平滑肌作用[J].上海医科大学学报,1990,17(5):350-354.
[9]王珏英,邵以德,张渊博,等.β-苯基乳酸对心血管系统的实验研究[J].上海医科大学学报,1991,18(4):295-297.
[10]程彰华,楼亚平,戴华娟,等.丹参素对微循环障碍和血浆乳酸含量影响的实验研究[J].上海医科大学学报,1987,14(1):25-29.
[11] HASHIMOTO Y,KOBAYASHI E,ENDO T. 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.
[12]孟琳,白云平,葛双启.果酸在化妆品中的应用[J].化学与生物工程,2005,22(4):45-46.
[13]贾江花.乳酸脱氢酶的克隆表达及酶学性质的研究[D].无锡:江南大学,2009:25-26.
[14] RODRIGUEZ N,SALGADO JM,CORTES S. Antimicrobial activity of D-3-phenyllactic acid produced by fedbatch process against Salmonella enterica[J]. Food Control,2012,25(1):274-284.
[15]赵明月,郑兆娟.重组大肠杆菌全细胞催化合成D-苯基乳酸的研究[J].广东化工,2015,42(6):29-30.
[16]黄志华,刘铭,王宝光,等.甲酸脱氢酶用于辅酶NADH再生的研究进展[J].过程工程学报,2006,6(6):1 011-1017.