| Cutinase?EC 3.1.1.74?is a multifunctional enzyme capable of hydrolyzing cutin and other various polyesters,triglycerides and soluble esters.It has a superior ability in hydrolyze polyester,and a broad application prospects.In the papermaking process,the problem of stickies affects the normal operation of papermaking equipment,resulting in a decline in paper quality.Analysis of the composition of the stickies showed that a large amount of synthetic polyesters was accounted.However,in the current study,the use of cutinase to hydrolyze polyesters for the treatment of stickies has not yet begun.In this study,cutinase from Humicola insolens,Fusarium solani pisi and Thermobifida fusca were selected,the cutinase function in hydrolyzing model substrate and practical application were characterized.The cutinase with better application performance was fermented and functionally optimized.The main findings are as follows:?1?H.insolens and F.solani pisi cutinase are expressed in Pichia pastoris.Using Pichia pastoris KM71 as the host strain,the effects of different expression vectors pPIC9K and pPICZ?A on H.insolens and F.solani cutinase expression were investigated.The activity of recombinant cutinase was up to 189 U·mL-1 and 256 U·mL-1,respectively.The optimum pH of the cutinase from both sources is 8.5,and the pH stability range is between 7.0-9.0.The optimum temperature was 80?and 40?,respectively.The half-life of F.solani cutinase at40?was 85 h.The half-life of H.insolens cutinase at 80?is 30 min.?2?The function of the cutinase hydrolysis model substance,poly?vinyl acetate?,poly?methyl acrylate?and poly?ethyl acrylate?,were characterized.After the suspension of the model substance was hydrolyzed by the cutinase,the decrease of turbidity was reduced from43.3%before the treatment to about 1%,and no precipitation occurred.When the turbidity reduction of the suspension is less than 1.5%,the amount of T.fusca cutinase added is about30%-40%and 8.3%-10%of the amount of H.insolens cutinase and F.solani cutinase.At the same time,the particle size of the suspension decreased by 14.4-37.5 nm,and the zeta potential increased by 0.67-1.37 mV.The effect of hydrolysis of poly?methyl acrylate?and poly?ethyl acrylate?by different sources of cutinase was also compared by pH-stat method.The results showed that the formation of formate ion and acetate ion in the suspension was highest when hydrolyzed by T.fusca cutinase for 12 h,which were 2.275 and 3.35?mol·mL-1,respectively.?3?The cutinase function in waste paper deinking and white water treatment was characterized.The brightness of paper can reach 41.62,41.81 and 42.01%ISO respectively after F.solani,H.insolens and T.fusca cutinase deinking without losing the mechanical strength of the paper.The paper brightness was higher than chemical and commercial lipase deinking paper brightness?36.49,39.6%ISO?.After the cutinase treatment of white water,the stability of the water sample increased.T.fusca cutinase had the best effect.After treatment at35°C for 12 h,the particle size of T.fusca cutinase treated water sample decreased by 36.7nm and zeta potential increased by 1.02 mV.?4?The fermentation of T.fusca and H.insolens cutinase was optimized.The fermentation of recombinant T.fusca cutinase constructed in the previous study was optimized.When the dry weight?DCW?of E.coli reached 13 g·L-1,IPTG with a final concentration of 25.0?M was added with a constant rate addition of lactose at a rate of 0.5g·L-1 h-1,the activity of extracellular T.fusca cutinase reached 2258.5 U·mL-1.Then,the high efficient fermentation of recombinant H.insolens cutinase was studied.The results showed that at the induction temperature of 30?,the initial induced bacteria concentration OD600was 100,and the methanol concentration was 1%.When sorbitol was added as co-carbon source with a ratio of 1:4 in both growing and induction periods,the fermentation was carried out for 96 h,H.insolens cutinase activity reached 2660 U·mL-1.The production time of cutinase was shortened by 50 h,and the production efficiency was increased from 17.78U·mL-1 h-1 to 27.71 U·mL-1 h-1,which was increased by 1.56 times.?5?The function of H.insolens cutinase was optimized.In order ot remove the"gate"structure,H.insolens cutinase mutants L66A,I169A and L66A/I169A were constructed.The results of hydrolyzing poly?vinyl acetate?,poly?methyl acrylate?and poly?ethyl acrylate?showed that the three mutants could improve the cutinase ability of hydrolyzing polyester.Under the same reaction conditions,the mutant L66A\I169A had the best result,and the addition of enzyme could reduce 18%-45%.Formate ion and acetate ion produced by hydrolysis of polymethyl acrylate and polyethyl acrylate were increased by 21%and 30% respectively. |
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