Gene Identification, High-efficient Expression And Molecular Modification Of Thermobifida Fusca Cutinase

Cutinase is a kind of hydrolase capable catalyzing the cleavage of ester bonds of cutin to release fatty acid. They display hydrolytic activity not only toward cutin but also a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Except for hydrolytic activity, cutinase also shows synthetic activity and transester activity. Therefore cutinase has been evaluated as a versatile lipolytic enzyme used in food and chemical industry. Rcently, it was found that cutinase has potential use in cotton bioscouring and synthetic fibers modification. Cutinase is the important enzyme in textile industry clean production.Fungal cutinase has been extensively investigated for many years. However, because of its low expression level and poor stability, recombinant fungal cutinase has not been produced in industry. In contrast to fungal cutinase, bacterial cutinase is more efficient in catalysis and easy to homologous expression. But there are just a few reports of bacterial cutinases. Most critically, no cutinase open reading frame has been identified in bacteria, thus it is impossible to study it in depth.In present paper, the gene of bacterial cutinase from Thermobifida fusca has been identified and high-efficient expressed in E. coli. And the recombinant T. fusca cutinase has also been molecular modified. The main results as follows:1. The pNPB hydrolase was purified from cutin-induced culture supernatant of T. fusca through ammonium sulfate precipitation, hydrophobic interaction (Phenyl Sepharose) and anion exchange (DEAE Sepharose) chromatography. The purified enzyme exhibited a high specific activity of 398.6 units/mg for pNPB. The purification fold is 7.6 and recovery yield is 9.3%. F. solani pisi cutinase was purified from supernatant of recombinant Bacillus subtilis through ammonium sulfate precipitation, hydrophobic interaction (Phenyl Sepharose) and cation exchange (CM Sepharose) chromatography. Two purified enzymes were used to hydrolyze apple cutin and the production were determined by GC/MS. It was found that both enzymes exhibited activity for cutin hydrolysis, yielding comparable fatty acid monomers.2. The purified T. fusca pNPB hydrolase was identified by Peptide Mass Fingerprinting Analysis, resulting in two significant matches, Tfu₀883 and Tfu₀882. The N-terminal amino acids were ANPYERGPN by sequencing. The gene encoding the mature form of Tfu₀882 and Tfu₀883 were amplified from T. fusca genomic DNA by PCR. The vector pET-20b(+) were chosen for expression, which contains a C-terminal His6-tag and a signal peptide PelB allowing the heterologously expressed proteins to be secreted. The constructed plasmid pET-20b/cutinase were expressed in E. coli BL21(DE3). The pNPB hydrolase activity of recombinant Tfu₀882 and Tfu₀883 in culture supernatant were 52.5 U/ml and 90.6 U/ml, which were 4.37 and 7.56-fold higher than that of cutin-induced T. fusca cells. The recombinant enzymes were purified to homogeneity in a single step by nickel affinity chromatography and exhibited a specific activity of 458.21 units/mg for Tfu₀883 and 223.13 units/mg for Tfu₀882. Both recombinant enzymes can hydrolyze cutin to release monomers. Tfu₀883 and Tfu₀882 is the encoding gene of T. fusca cutinase3. The effect of IPTG induction concentration and temperature on the production of recombinant Tfu₀883 were determined. It was found that without IPTG induction and culture cell at 25°C for 80 h, the pNPB hydrolase activity in the culture supernatant was 130.52 U/ml. In addition, there was a lot amount of recombinant protein accumulated in periplasm. So that the transformation of recombinant protein out of outer membrane became the limiting-step of secretion. Adding additives and changing the environment condition to accelerate the secretion of recombinant protein is effective. The study showed that, adding 100 mM glycine and 1mM TDOC at mid-log growth phase could advance the peak of activity from 80 h to 54 h. Culturing the cell at 25°C until late-log growth phase, then increased the temperature to 37°C, in this way, the peak of activity can be advanced from 80 h to 62 h.4. Recombinant Tfu₀882, Tfu₀883 and F. solani pisi cutinase were all determined to be monomeric in solution. Tfu₀882 and Tfu₀883 exhibited an optimal temperature at 60°C and superior thermostability, while F. solani pisi cutinase displayed an optimal temperature at 40°C and significantly less stable. Furthermore, all three enzymes exhibited a pH optimum of about 8.0 and pH stability range is 6.0-9.0. Three enzymes can hydrolyze soluble synthetic esters, insoluble triglycerides and polyesters. They all prefered short chain substrate and showed no substrate position specificity. F. solani pisi cutinase exhibiting the highest affinity and the highest catalytic efficiency for pNPB. In addition, F. solani pisi cutinase exhibited the highest activity toward PET, while Tfu₀883 exhibited less activity. Three enzymes did not possess interfacial activation phenomenon, did not require divalent cations for activity. Mn active the three enzymes while Cr and Hg inhibited enzyme activity intensely. Triton X-100 and Tween 20 inhibited the activity of Tfu₀882 and Tfu₀883 while SDS inhibited all the three enzymes in competitive way. TDOC active the F. solani pisi cutinase greatly. Tfu₀882 and Tfu₀883 demonstrated great stability in many organic solvents such as mathol and ethnol while F. solani pisi cutinase is less stable in organic solvents.5. A homology model of Tfu₀882 and Tfu₀883 based on Streptomyces exfoliates lipase structure was constructed by the SWISS-MODEL homology-modeling web server. The predicted structure exhibits anα/βhydrolase fold of both two enzymes. In addition, it revealed a S170-H248-D216 catalytic triad for Tfu₀883 and S188- H266-D234 catalytic triad for Tfu₀882. Both two enzymes do not contain a lid insertion which resulting in a nucleophilic serine exposed to the solvent. The Tfu₀882 and Tfu₀883 genes are sequential in the genome and belong to separate operons. There is no synergism between the two enzymes in the degradation of cutin. Based on the structure model and rational analysis, the mutation enzymes Tfu₀883-I218A, Tfu₀883-W195A/F249A and Tfu₀883-Q132A/T101A were constructed. The study of catalysis properties showed that mutants exhibited no significant effect on low molecular weigh substrate. Mutants Tfu₀883-I218A and Tfu₀883-Q132A/T101A showed higher ability in hydrolyzing cutin. Tfu₀883-Q132A/ T101A exhibited obviously higher hydrolysis ability on PET polymer.