Construction Of Alkaline Pectate Lyase PEL168 High-yield Yeast And Molecular Modification

The applications of pectate lyase in textile industry are promising,mainly in the ramie degumming and pre-treatment of fabric bioscouring.Compared to the conventional processes which involve high alkaline and high temperature treatment,the enzyme based treatments have significant advantages in fibers protectiveness,improved efficiency of refining,reducing energy consumption and pollution,and more environmental friendly.It's highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost.In the previous study,the alkaline pectate lyase gene pel came from Bacillus subtilis 168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905 BDM.To improve the expression level,a mass of recombinant plasmid pHBM905BDM-pels were electroporation the P.pastoris GS115.Obtained recombinants through HIS4 selection marker and the transformants were further screened on pectin plates,where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/m L.Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells after linearized with Sac I.Since the transformants with more copies of inserted fragments,the capacity of G418 antibiotic resistance was stronger theoretically.Subsequently,high-copy transformant was screened by using the medium containing 4 mg/m L antibiotics G418,strain GS115-pHBM905BDM-pPIC9K-pels1 was identified which showed the increased activity of 770 U/m L and the copy number of pels was 7 confirmed by qRT-PCR.Finally,the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1 reached to 2271 U/mL in 5 L fermentor.In the previous study,the alkaline pectate lyase gene pel has been expressed in Escherichia coli also and displayed specific activity of 353 U/mg and lost 50% of its initial activity after incubation at 50°C for 2 h.Since the the tertiary structure of PEL168 was resolved,some rational design were done based on the structure and selected mutation sites.Fortunately,a mutant V132 F with 1.32-fold higher specific activity and improved pH stability was obtained.Objective of this study was to apply different designs to create mutants to increase its specific activity and thermo-stability,so efficiently being applied in bioscouring.1)Initially,rational design was applied to generate a mutant B170/173 C which would generate a new disulfide bond to stable the locality structure,however lost activity completely.2)Meanwhile,a random mutagenesis library was constructed based on the wild-type PEL168 through error-prone PCR and a mutant K47E(640 U/mg)with higher specific activity and thermo-stability was obtained.3)Then,the double mutant K47E/V132 F was created to explore the increased activity,which displayed 2.21-fold specific activity compared to PEL168.4)Since K47 site played much role on activity,K47D/V132 F was screened by site-saturation mutagenesis,which showed 3.93-fold improved specific activity compared to PEL168.5)Furthermore,by sequence alignment,the mutant K47D/V132F/R272 W exhibited a 4.37-fold higher specific activity compared to PEL168 and the half-life of inactivation at 50 °C for this mutant extended to over 5.6 h.Kinetic parameter analysis revealed that the catalysis efficiency of the mutant K47D/V132F/R272 W was higher than that of the PEL168.Structure analysis showed that the catalytic triad of mutant K47D/V132F/R272 W has a larger spatial distance and more stable structure,which contribute the improved specific activity.Increased hydrogen bonds and decreased free energy contributed to the improved thermo-stability.The mutant K47D/V132F/R272 W displayed well ability in bioscouring of gray cloth and the fibers became more smoothly after treated with enzyme when observed in scanning electron microscope.In conclusion,a more efficient mutant K47D/V132F/R272 W was screened firstly in this report for potential applications in bioscouring in the textile industry.