| Polyester fiber is widely used as a textile material due to its various advantages like good elasticity,excellent wear resistance,high strength,fold resistance and fast drying,and becomes one of the fastest growing synthetic fibers in the textile industry.PET fiber is a polyester material formed by the dehydration polymerization of terephthalic acid and ethylene glycol.It has strong hydrophobicity and is not easy to dye,which seriously affects the quality of textiles.The cutinase derived from Thermobifida fusca can hydrolyze the ester bond in the PET fiber and produce hydrophilic-COOH and-OH groups to increase its hydrophilicity.However,PET is highly crystalline,which reduced the accessibility of cutinase to the polyester chain in PET fiber and seriously affects the catalytic efficiency of cutinase.Therefore,the enzyme treatment can be carried out at close to or higher than the glass transition temperature of PET?69-80??.Yet,the thermal stability of T.fusca cutinase is poor under this temperature condition and needs to be improved by molecular modification so as to meet the application requirements.In this study,we used the plasmid p ET-20b?+?-cut as a template,through rational design,introduced disulfide bonds in a site-directed mutation manner,and obtained a strain T.fusca cutinase mutant D204C/E253C whose thermal stability and pH stability were significantly improved and other excellent traits remained unchanged.And we achieved efficient soluble expression of mutant D204C/E253C through mutation optimization,co-expression and no signal peptide.Compared with wild-type T.fusca cutinase,using the mutant D204C/E253C to modify the PET fiber hydrophilism at 80?can significantly improve the efficiency.The main findings are as follows:?1?The rational design of disulfide bonds improves the thermal stability of T.fusca cutinases:using plasmid pET-20b?+?-cut as a template,through rational design,disulfide bonds were introduced in the way of site-directed mutation to obtain 11 mutants.Studies on the thermal stability of the mutant showed that the thermal stability of the mutant D204C/E253C was significantly improved.Compared to the wild-type T.fusca cutinase,which retained only 10.7%of its activity after being incubated at 70?for 10 minutes,the mutant D204C/E253C did not experience any decrease in enzyme activity under this condition,and its half-life at 80?could reach 16 h,even incubated at 90?for 10 min,still have 55.6%activity.Purified wild-type T.fusca cutinase and mutant D204C/E253C,comparing the enzymatic properties of the two,the results indicated that the mutant D204C/E253C pH stability was also significantly improved except for its enhanced thermal stability.?2?Efficient soluble expression of mutant D204C/E253C:?a?By optimized mutation based on pET-20b?+?-D204C/E253C,the enzyme activity increased 3.89 times which is62.5%of E.coli BL21?DE3?/pET-20b?+?-cut;?b?By constructing the recombinant strain E.coli Origami B?DE3?/pSCDsbC-D204C/E253C,the mutant D204C/E253C and the disulfide isomerase Dsb C in E.coli Origami B mutant DE3 co-expression,so that the mutant D204C/E253C can perform normal soluble expression.The extracellular enzyme activity reaches 61 U·mL-1,which is 1.09 times of E.coli BL21?DE3?/pET-20b?+?-cut;?c?The recombinant strain E.coli BL21?DE3?/pET-24a?+?-D204C/E253C was constructed to remove the signal peptide during cutinase expression,which significantly increased its soluble expression.The extracellular enzyme activity reached 192 U·mL-1 and is 3.43 times of E.coli BL21?DE3?/pET-20b?+?-cut;when the induction time is extended to 48 h,the extracellular enzyme activity can reach 351 U·mL-1,which is 6.26 times of E.coli BL21?DE3?/pET-20b?+?-cut.?3?Fermentation preparation of mutant D204C/E253C and its application in the hydrophilic modification of PET fiber:the mutant D204C/E253C was subjected to 3-L tank high-density fermentation,and the whole cell enzyme activity can reach up to 1449 U·mL-1.The PET fiber of mutant D204C/E253C treated at 80?had a wetting time reduced to 6.3 s,which was 1.62 times higher than the PET fiber treated by wild-type T.fusca cutinase at 50?.Further SEM analysis showed that the etch marks on the surface of PET fiber treated by mutant D204C/E253C at 80?were more and more obvious than those of wild-type T.fusca cutinase treated at 50?. |
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