Construction Of Pichia Pastoris Engineering Strain For Degumming Pineapple Leaf Fiber And Its Expression,application

The study on pineapple leaf fiber as a new type of textile materials degumming has aroused attention. Pectin lyase with the role of pectin degradation and xylanase can be degradation of hemicellulose, the main component of xylan. Pectin lyase and xylan play key role of pineapple leaf fiber degumming. The expression of recombinant pectin lyase has been accomplished in E.coli. and have been preserved pPIC9K-PelC and a strain named P2 which could high yield xylanase in our previous work. The xylanase gene fragment xyn2 was successfully amplified from mRNA of Trichoderma viride (AS3.3711) by RT-PCR (reverse-transcription PCR assay) technique, and then was ligased with pPIC9K vector to construct the expression vector pPIC9K-xyn2.The expression vector pPIC9K-xyn2 was electric of strain Pichia pastoris GS115, then pPIC9K-xyn2 and pPIC9K-PelC were electric of strain Pichia pastoris GS115. After screening on his-deficient plates,YPD-G418 plates and methanol induced expression procedures sequentially, a strain named X5 which could high yield xylanase and another strain named PX6 which could high co-express xylanase and pectin lyase were obtained respectively.The product of P2,X5,PX6 were analysed by SDS-PAGE. The results demonstrated that xylanase was secreted by X5,PX6 and pectin lyase was secreted by P2,PX6, of which the molecular weight was 21KD and 43 KD respectively.The recombinase enzymatic properties of P2,X5 and PX6 were analyzed. The result indicates that the highest activity of pectin lyase secreted by P2, xylanase by X5, pectin lyase by PX6 and xylanase by PX6 was 14.2 U/ml,5.8 U/ml,12.6 U/ml and 4.2 U/ml respectively; The optimum pH and temperature of pectin lyase were pH 5.4 and 50℃, respectively; The optimum pH and temperature of xylanase were pH 6.0 and 50℃, respectively.Pectin lyase and xylanase with high enzymatic activity from a strain screened by PX6.The orthogonal experiments were carried out to establish the optimal degumming process, processing at 50℃for 4 h with pectin lyase and xylanase 12% and pH5.5. The results of degumming demonstrated that the pineapple leaf fiber yield was favored, especially pectin degradation fulfil textile technology requirements, but the content of semi-fiber was sighty higher. The fineness of fiber was favored and smaller and fracture strength was decreased.