| Cutinase(EC3.1.1.74)is a multifunctional hydrolase,which can hydrolyze short chain or long chain aliphatic ester and triglyceride,also can be used to degrade the polymer cutin of plant.It is an important enzyme in the bioscouring industry of cotton fiber.In this study,the recombinant bacteria of Humicola insolens(H.insolens)cutinase gene(hic)in Escherichia coli(E.coli)was constructed to study its enzymatic properties.Additionally,fermentation of recombinant bacteria in 3-L NBS 115 Bioflo was further studied and optimized.Finally,the recombinant enzyme was used in biscouring of cotton fiber.The main results are as follows:1.Cloning and expression of H.insolens cuntinase in E.coli has been achieved for the first time.The recombinant strain E.coli BL21(DE3)/pET-20b(+)/hic was constructed.The recombinant strain was cultured in shake flask for 24 h,activity of cutinase in the supernatant of fermentation broth was 170 U mL-1.Then recombinant cutinase was purified by ammonium sulfate sediment and cation exchange column,its enzymatic properties were investigated.The results showed that the optimum reaction temperature of the recombinant enzyme was 80 ?and the optimum reaction pH was 8.5.The half-life time of the recombinant enzyme was 11 min under the condition of 80? and pH 8.5,the half-life of the recombinant enzyme was 14 h under the condition of 60? and pH 8.5,and it could remain stable for a long time under the conditions of 4? with pH 5.0-10.0.The specific activity of recombinant enzyme was 1047 U· mg-1.2.At the level of 3-L fermentation,a new strategy was designed which induced with high cell concentration and high-intensity to further improve the expression level of recombinant protein expression in 3-L fermentation tank.The final fermentation conditions were as follows:at initial stage,cultured the recombinant strain E.coli BL21(DE3)/pET-20b(+)/hic at 37?and pH 7.0.When cell concentration up to 75,cooling the temperature to 30?,and adding lactose solution with constant velocity of 0.8 g·L-1·h-1 for 8h,the activity of the recombinant enzyme reached 4778 U mL-1,which was 28 times about the level of shake flask.3.The efficient of recombinant enzyme in biscouring of cotton fiber was studied and optimized.Two ways of using H.insolens cutinase were pointed.First,cotton fiber treated solely by H.insolens cutinase.The optimum conditions were as follows:the reaction temperature was 80?,reaction pH was 8.5,adding 1g cotton fiber with 800U H.insoelns cutinase.Total time of reaction was 60 min.The wetting time of treated cotton was 1.5 s,its dyeing uptake ratio reached 87%,K/S value was 11.2,and weightlessness rate was 4%.Second,cotton fiber treated by H.insolens cutinase and T.fusca cutinase.The best conditions were as follows.Using 1g raw cotton fiber as substrate and 20mL pH 8.5 Tris-HCl buffer as solution.Adding 200 U·g-1 substrate H.insolens cutinase reacted in 80? for 5min.Cooling to 60? and adding 300 U·g-1 substrate T.fusca cutinase at the same time.Total amount of cutinase was 500 U g-1 substrate,total reaction time was 70 min.The wetting time of treated cotton was 5 s,the dyeing uptake ratio was 84%,K/S value was 11,and weightlessness rate was 4%.Single enzyme and double enzyme treatment process with H.insolens cutinase could simplify the process of treament,reduce enzyme adding amount and avoid those disadvantage caused by chemical method. |
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