摘要
以表达α-酯酰基转移酶的重组大肠杆菌作为全细胞催化剂,连接丙氨酸甲酯盐酸盐与谷氨酰胺合成丙谷二肽,是目前生产丙谷二肽最理想的方法。利用高密度发酵法,能提高微生物的生产量,降低二肽生产成本。首先,在摇瓶水平下,利用响应面模型对于基础培养基碳源、氮源、无机盐等因素进行研究,在此基础上,对补料培养的重要因素如补料种类、补料方式等进行研究。最后,利用高密度培养的重组大肠杆菌作为全细胞催化剂进行丙谷二肽的合成。确定了最佳半合成培养基的组成为:葡萄糖质量浓度为10g/L、混合氮源质量浓度为24g/L、KH2PO4质量浓度为4.62g/L、K2HPO4质量浓度为25.08g/L;确定了溶氧反馈补料的最佳补料控制条件,在30h内,菌体OD620值最高达到了67,是优化前摇瓶培养的14倍。丙谷二肽的质量浓度达到34.56g/L,生产效率约为7g/(L·min)。研究为高效低成本生产丙谷二肽奠定了基础。
L-alanyl-L-glutamine (Ala-Gln) is an important functional dipeptide, which has important value in food, medicine and health care products. Using recombinant Escherichia coli to express α-amino acid ester acyltransferase as whole-cell catalyst, which linked alanine methyl ester hydrochloride with glutamine to synthesize Ala-Gln, was an ideal method for the production of Ala-Gln. The high-cell-density cultivation could increase the proliferation concentration of microorganisms and reduce the cost. Firstly, the medium of E. coli BL21-pet29a- SsAET strain which expressing α-amino acid ester acyltransferase was optimized using response surface methodology based on the basic culture medium composition, such as carbon source, nitrogen source, and inorganic salt in flask-shaking experiment. Then, the important factors for fed-batch cultivation, such as pH, feeding medium and feeding mode, were investigated in fermenter. Finally, the recombinant E. coli by the high-density cultivation as whole-cell catalyst were used for the synthesis of dipeptide. The results showed that the optimum concentration of the semi-synthetic medium was: glucose 10 g/L, mixed nitrogen source 24 g/L, KH2PO4 4.62 g/L and K2HPO425.08 g/L. And the optimum feeding method was controlled using the automatic feedback by dissolved oxygen against pH-controlled. When the semi-synthetic medium was used with automatic feedback by dissolved oxygen, the OD620 value of the bacteria reached up to 67 at 30 h, which was 14 times better than that of flask culture. When the high density-culture of recombinant E. coli was used to synthesize dipeptide, the concentration of Ala-Gln reached 34.56g /L and the production efficiency was 7 g/(L·min), which could further enhance its potential for industrial applications.
作者
裴绪泽
李益民
杜聪
袁文杰
PEI Xuze;LI Yimin;DU Cong;YUAN Wenjie(School of Biotechnology,Dalian University of Technology,Dalian 116024,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2020年第13期30-35,共6页
Food and Fermentation Industries
关键词
丙谷二肽
转酯酶
高密度发酵
过程控制
优化设计
L-alanyl-L-glutamine
ester acyltransferase
high cell density fermentation
process control
optimal design
