Expression, Codon Optimization, Characterization And Application Of An Alkaline Pectate Lyase From Bacillus Subtilis168

Pectate lyases can catalyze dissimilation of the a-1,4-glucosidic bond of pectin polymer to produce oligosaccharides, and they have potential utility in animal-feed preparation, fruit wine clarification, spinning, and paper production, especially in enzymatic degumming. The conventional degumming process is performed with alkaline treatment at high temperature, which causes serious environmental pollution, energy consumption and fiber damage. Natural fiber of ramie contains gum-like material that can be removed by pectate lyase and other enzymes so that the use of harsh chemicals is reduced significantly. Hence, enzymatic degumming can not only decrease environmental pollution and energy consumption but can also improve yield and quality of degummed ramie. For industrial application of enzymatic degumming, it is important to produce cheap, effective and cellulase-free pectate lyase.A pectate lyase gene(pel168) from Bacillus subtilis was expressed in Escherichia coli and Pichia pastoris, respectively. Recombinant pel168expressed in E. coli (PEL168E) was the same as predicted with a band of46kDa, whereas the enzyme expressed in P. pastoris (PEL168P) had two bands of48.6kDa and51.4kDa. Deglycosylation digestion showed that PEL168P was glycosylated. The optimal reaction temperature of the two PEL168S was50℃, and the optimal pH was9.5. After preincubation at60℃for20min, PEL168E completely lost its activity, whereas PEL168P kept26%residual activity. PEL168P had a specific activity of1320U/mg with a Km of0.09mg/ml and a Vmax of18.13μmol/min. K+, Li+, Ni2+and Sr2+showed little or no inhibitory effect on PEL168P activity; Mn2+, Hg2+and Cu2+inhibited the most enzyme activity; and Zn2+, Fe3+, Mg2+and Co2+reduced enzyme activity by35-55%. However, the enzyme activity was enhanced by about38%by Ca2+Codon optimization was applied to pel168of Bacillus subtilis to enhance its expression levels in P. pastoris.294nucleotides were mutated, consistent with the codon bias of P. pastoris. When compared with the originally cloned pel168gene of Bacillus subtilis, the synthetic codon optimized pel168gene was expressed at a higher level in P. pastoris (PEL168S). Batch fermentations of both clones with the same copy number under controlled conditions, indicated that codon optimized PEL168S enzyme activity increased to1.52fold after72h of methanol induction, and thus is a low-cost alternative for the spining industry. Same of the high copy P. pastoris transformants, which was examined by Realtime-PCR, the highest had11copy quantities of pel168s gene. In addition, we also determine the application parameters of PEL168P in ramine degumming and bio-scouring, the results show that the alkaline pectase lyase played an important role in the environmental protection production of the textile industry.