Recombinant Expression,Fermentation Optimization Of Pullulanase And Its Application In Trehalose Production

Pullulanase is a debranching enzyme that specifically hydrolyzed a-1,6 glycosidic bond in polymers such as starch and pullulan.Pullulanase can improve substrate utilization and reduce product costs in the starch processing industry.In previous study,trehalose was prepared using starch as a substrate and using maltosyl trehalose synthase(MTSase)and maltosyl trehalose hydrolase(MTHase)from Arthrobacter ramosus S34.However neither of these enzymes can hydrolyze the ?-1,6 glycosidic bond in the substrate.Therefore,the addition of pullulanase,whose properties meet with these two enzymes(temperature 45?,pH 5.5).can significantly improve the utilization of the starch and the conversion rate of trehalose.Bacillus acidopullulyticus pullulanase(BapulA)and Bacillus naganoensis pullulanase(BnpulB)are consistent with the two enzyme properties and can be used for the preparation of trehalose.In this study,pullulanase BapulA and BnpulB were expressed in Escherichia coli and Bacillus subtilis,respectively,and the fermentation conditions of the recombinant bacteria and the influencing factors in the conversion of trehalase were optimized.The main findings of the thesis are as follows:(1)After the codon optimization of the gene BapulA from Bacillus acidopullulyticus was artificially synthesized,the recombinant plasmid pET-20b(+)-BapulA was obtained.Then the plasmid was transformed into E.coli,resulting E.coliBL21(DE3)/pET-20b(+)-BapulA.Fermentation in shake flasks,the highest enzyme activity reached 201.0 U·mL-1.The optimum fermentation conditions at 3 L fermentor level were stated as follows:the growth temperature and pH of the pre-induction were 30? and 7.0,respectively.When the cell density reached a certain concentration(OD600=50),the fermentation temperature was shifted to 25?,and the pH was adjusted to 6.2,lactose flow rates was 0.4 g·L-1·h-1.During the fermentation process,glycine was added at the concentration of 1.5 g·L-1 when the OD600 reached 15,45 and 75,respectively.When OD600 reached 105,glycine was added at a concentration of 3 g·L-1.The optimal total pullulanase activity was 1910.1 U·mL-1.(2)The recombinant plasmid pHY300PLK-BapulA was constructed.Then the plasmid was transformed into B.subtilis CCTCC M 2016536 by electric shock to construct a recombinant strain B.subtilis CCTCC M 2016536/pHY300PLK-BapulA.The strain was subjected to shake flask fermentation and the enzyme activity was 0.8 U·mL-1.In addition,the culture conditions of the recombinant bacteria were optimized,and the highest enzyme activity was 23.4 U·mL-1,which was 28 times higher than that of no optimization.(3)After the codon optimization of the gene BnpulB from Bacillus naganoensis was artificially synthesized,the recombinant plasmid pET-22b(+)-BnpulB was obtained.Then the plasmid was transformed into E.coli resulting E.coliBL21(DE3)/pET-22b(+)-BnpulB.The fermentation was carried out in a shake flask,and the highest enzyme activity reached 568.5 U·mL-1.(4)The recombinant plasmid pHY300PLK-BnpulB was constructed,then transformed into B.subtilis CCTCC M 2016536 to obtain the recombinant strain B.Subtilis CCTCC M 2016536/pHY300PLK-BnpulB.Results of shake flask showed that the highest enzyme activity of the fermentation was 80.4 U·mL-1.In the shake flask,the carbon source and nitrogen source of the fermentation medium were optimized.The best carbon source was glucose,and the best nitrogen source was yeast extract powder and NH4H2PO4.Furthermore,the feeding medium was optimized at the 3 L fermenter level,when 400 g·L-1 glucose as carbon sourse and the 100 g·L-1 yeast extract powder as the nitrogen source,the maximum enzyme activity was 2361.1 U·mL-1,which was 29 times than the enzyme activity of shake flasks.(5)Using starch as the initial substrate,production of trehalose via pullulanase with MTSase and MTHase were studied.The temperature during the enzyme conversion,initial pH,and substrate concentration,and the amount of pullulanase were investigated.The optimal reaction temperature was 45?,the optimal initial pH was 6.0,the optimal substrate concentration was 15%.The enzyme amount of BapulA was 15 U·g-1 starch,the substrate conversion rate was 66.7%,the optimal enzyme addition amount of BnpulB was 2 U g'1 starch,and the substrate conversion rate was 67.9%.