| ?-Poly-L-lysine(?-PL)is a homopoly(amino acid)generally composed of 25–35L-lysine residuals linked by?-amino and?-carboxyl groups.?-PL as a new biologial food preservative has been approved in Japan,United States,South Korea,China and other countries.?-PL is mainly produced by S.albulus through aerobic fermentation,but some weakness in the fermentation process such as:low substrate conversion rate(<10%),long fermentation period(7-8 days)and high oxygen consumption(>1.0vvm).Those weakness cause the high cost of?-PL and become the main barrier limiting its wide application.Therefore,it is of great value to explore new production methods.In this thesis,S.albulus M-Z18 was used as the research object,and the system of producing?-PL from precursor L-lysine by S.albulus M-Z18 was constructed and optimized.Then,the physiological function of the degrading enzyme was clarified by studying the physiological function of the degrading enzyme.Finally,the efficiency of producing?-PL from precursor L-lysine with S.albulus M-Z18 was improved by overexpression the?-PL synthetic enzyme(Pls).The main research contents and results are as follows:(1)The system that synthesis?-PL from precursor L-lysine by S.albulus M-Z18 has been constructed and optimized.We established optimal conversion conditions by optimizing the system p H,temperature,glucose concentration,wet cell concentration,substrate concentration,cell culture time and permeability agent type and concentration.The optimal conditions were as follows:p H 4.0,temperature 30?,culture time 12 h,wet cell concentration 450 g·L-1,glucose concentration 60 g·L-1 and L-Lysine concentration 6g·L-1.The yield of?-PL reached 8.2 g·L-1 which 5.48 times as much as shake flask fermentation after 96 h conversion.(2)Research on the conversion of degradation pathway knockout strains and the physiological function of?-PL degrading enzyme(Pld).Firstly,the knockout,complementation,and overexpression mutants of pld were constructed and the degradation activity of?-PL by mutants were analyzed.It was found that all mutants were successfully constructed.Compared with the S.albulus M-Z18,the?-PL conversion production of?pld II,pld I?pld II decreased by 69.2%and 66.5%respectively,but?pld I and OE-pld Ipld II were the same as the original strain.At last,the reason for the abnormal conversion of knockout strains were explained from the physiological function of Pld.The existence of Pld can help the strain to avoid the harmness from?-PL,and the degradation activity of pld II is higher than pld I.Therefore,Without Pld the?-PL will damage the strain and resulting in the increase of p H and the decrease of yield finally.(3)Overexpression?-PL synthetase(Pls)improve the efficiency of transforming precursor L-lysine to?-PL.Two constitutive strong promoters Kas OP*and Perm E*were used to overexpress pls.The results showed that the production of?-PL of OE-Kas OP*-pls and OE-Perm E*-pls was 1.67 g·L-1and 1.72 g·L-1 respectively after 72 h flask fermentation,which was 26%and 29.5%higher than S.albulus M-Z18 respectively.The conversion production of OE-Perm E*-pls increased by 11.1%compared with S.albulus M-Z18.However,the yield of OE-Kas OP*-pls did not increase significantly.After 96 h conversion in 1 L fermentor,the yield of OE-Kas OP*-pls and OE-Perm E*-pls were 13.5 g·L-1 and 14.29 g·L-1respectively,which increased by 27.96%and 35.45%compared with S.albulus M-Z18.The polymerization of?-PL synthesized from the precursor L-lysine was highly consistent with the fermentation method. |
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