Construction And Characterization Of Thermobifida Fusca Cutinase-CBM Fusion Proteins And Their Application In Textile Industry

Cutinase is a multi-functional esterase of degrading not only cutin component of plant cuticle, but also other long chain or short chain of fatty acid ester, emulsion triglycerides and a variety of natural or synthesis ester, etc. Recently, it was reported that cutinase has potential use in cotton bioscouring and surface modification of synthetic fiber, which was the new direction of application for cutinase in the textile industry. Thus, study of cutinase can promote the development of eco-textile industry, which allows energy saving, disposal reduction, and environmentally friendly technologies.In recent 30 years, fungal and bacterial cutinase has been extensively studied. However, it has not produced enzymes having high catalysis, wide applicability and thermally stable in textile industry. The main reason is the low affinity between the enzyme and fiber substrate, which greatly reduced the hydrolytic effect. Previously, site-directed mutagenesis approach has been performed to replace the specific amino acid residue near the active site of enzyme to improve its hydrolytic activity toward polyester. However, this method is of heavy workload, slow effective and little successful probability. By analysis of the surface structure of cotton fiber, there are rich in cellulose and hemicellulose components besides cutin, wax, pectin and other impurities. Thus, cellulose-binding module （CBM） for high adsorption on cellulose, could be fused to cutinase to construct fusion enzyme, which was used in biological treatment of textile fiber.In this paper, two T. fusca cutinase-CBM fusion genes have been constructed and over-expressed in E. coli. The potential use of cutinase-CBM fusion proteins in bioscouring and surface modification of synthetic fiber has been discussed. Also the molecular modification of fusion protein has been performed. The main results as follows:（1） Considering the condition of the textile fiber process, the cutinase-CBM fusion proteins were generated by fusing either the CBM of cellulase Cel6A from T. fusca (CBM_Cel6A) or the CBM of cellulase CenA from C. fimi (CBM_CenA) to the C terminus of T. fusca cutinase through overlapping PCR amplification. The fused genes were subsequently inserted into the expression vector pET-20b（+）, which encodes a C-terminal His6 tag and an N-terminal signal peptide PelB to allow the expressed proteins to be secreted. The resulting constructs pET20b/cutinase-CBM_Cel6A and pET20b/cutinase-CBM_CenA were used for protein expression in E. coli BL21（DE3）. Both fusion enzymes were purified by ammonium sulfate fraction and Ni-Sepharose affinity chromatography. SDS-PAGE results demonstrated that they were purified to homogeneity with the same molecular mass of ⁴5 kDa. In addition, the purified enzymes were active with specific activities of 440 U/mg for cutinase-CBM_Cel6A and 337 U/mg protein for cutinase-CBM_CenA. The results showed that both cutinase-CBM_Cel6A and cutinase-CBM_CenA exhibited an optimal temperature at 50°C, whereas the native cutinase displayed an optimal temperature at 60°C. The half-life of the native cutinase was 70 h at 50°C, while those of cutinase-CBM fusion enzymes were both 53 h at 50°C. All native and fusion enzymes exhibited the same optimal pH of 8. Similar pH stabilities at a pH range of 6 to 9 were observed for all three enzymes. Three enzymes can hydrolyze soluble esters （pNPB）, insoluble triglyceride and cutin. Their K_m values were similar to that of the native cutinase, while their catalytic efficiencies (K_cat/K_m) were 94% (for cutinase-CBM_Cel6A) and 85% (for cutinase-CBM_CenA) of that of the native cutinase.（2） The effect of IPTG and lactose induction concentration on enzyme production was determined. The optimal concentration of IPTG was 0.4-0.6 mmol/L, the optimal concentration of lactose was 2 g/L. The effect of glycine on the enzyme production was determined. It was found that the enzyme activities were increased by 1.5-1.6 fold by adding 0.5% of glycine in the logarithmic growth stage. The effect of surfactants of different concentrations on enzyme production was investigated. The results showed that 1% of Triton X-100 can obviously promote enzyme production by 1.8-2.1 fold. Tween 80 and Tween 60 have no obvious effect on enzyme production. JFC has severely inhibited cell growth and enzyme production. In the fed-batch culture in 3 L fermenter, the fermentation period was shorten by half than that in flask, and the maximum of activities were 286 U/mL for cutinase-CBM_Cel6A, 234 U/mL for cutinase-CBM_CenA and 506 U/mL for cutinase, which were all about 5-fold than that in flask. Our study provides a foundation for the further industrial production.（3） The results showed that, compared to T. fusca native cutinase, the binding of cutinase-CBM_Cel6A was enhanced by 2% in the absence of pectinase and 40% in the presence of pectinase, whereas the binding of cutinase-CBM_CenA was enhanced by 28% in the absence of pectinase and 45% in the presence of pectinase. Their hydrolytic efficiencies toward cotton fiber were also compared. In the absence of pectinase, the amount of released fatty acids was similar to that of native cutinase for cutinase-CBM_Cel6A but was 1.8-fold higher for cutinase-CBM_CenA. In the presence of pectinase, however, both fusion enzymes released almost the same amount of fatty acids and exhibited a catalytic efficiency 3-fold higher than that of the native cutinase. GC/MS was used to analyze the degradation of the cotton fabric cuticle; FT-IR microspectroscopy was used to study changes in the chemical composition of the cotton fabric epidermal layer; and SEM was used to monitor minor changes in the morphology of the fiber surface. Our results indicate that cutinase-CBMs in combination with pectinase have a greater effect on cotton fabric than does cutinase. Following scouring with cutinase-CBMs and pectinase, the performance of cotton fabric in terms of its wettability and dyeability is similar to that following alkali scouring.（4） The results showed that, compared to native cutinase, the binding activity was enhanced by 15% for cutinase-CBM_Cel6A and 16% for cutinase-CBM_CenA. Both cutinase-CBMs released almost the same amount of acetic acid, which was improved by 1.6- fold higher than native cutinase. These data demonstrated that bacterial native or fusion cutinase presented an activity with a great potential as acetyl esterase for cellulose acetates. The wetting time of enzymatic treated acetate fibers were 18 min for cutinase-CBM_Cel6A, 16 min for cutinase-CBM_CenA and 28 min for cutinase, which showed that cutinase-CBM fusion proteins caused a higher wettability than cutinase. The use of nonionic surfactant Triton X-100 could further improve enzymatic hydrolysis of cellulose acetate fiber in terms of wettability and dyeability. Cutinase was used to modify polyester fibers and terephthalic acid substance was detected by using UV analysis. Combined utilization of cutinase and Triton X-100 can significantly improve the hydrophilicity of polyester.（5） A homology model of C. fimi CBM_CenA based on C. fimi CBM_Cex structure （PDB 1EXG） was simulated by SWISS-MODEL web server. The model revealed that three highly conserved tryptophane resides were located at the same flat surface. Sequence comparison of family CBM2 from different sources showed that three tryptophan residues corresponding to Trp14, Trp34 and Trp50 of CBM_CenA are strictly conserved; a fourth tryptophan residue, corresponding to Trp68, is less conserved in members of family CBM2. Seven cutinase-CBM_CenA mutants were constructed by rational analysis. Enzyme characterization showed that they displayed similar thermostability and pH stabilities in response to the native enzyme. When compared to the native enzyme, the binding activity was enhanced by 14% for W68L and 17% for W68Y; the released amount of terephthalic acid was improved by 1.4-fold for W68L and 1.5-fold for W68Y. However, other mutants showed no increase in binding and hydrolytic activity toward PET fiber.