Characterization Of Hyperthermostable Recombinant Xylanase From Thermoascus Aurantiacus Expressed In Pichia Pastoris

Objectives To improve the thermostablility of xylanase from Thermoascus aurantiacus by heterologous expression in Pichia pastoris.Methods Optimization using the original bias thermophilic bacteria aurantiacus gene sequences of the xylanase gene xyn A(987bp)based Pichia codons.The recombinant plasmid p GAPZ?A-xyn A,p PIC9k-xyn A,after linearization electroporation transformed into the expression host Pichia pastoris GS115 construct recombinant strain.After screening with YPD agar plates containing zeocin,the positive transformants were screened by extraction of the genome.The bleomycin concentration gradient YPD agar plate was used to re-screen the transformants.The recombinant xylanase was isolated and purified by ammonium sulfate precipitation,organic solvent precipitation,ion exchange column chromatography and molecular sieve chromatography.The size of recombinant xylanase was determined by SDS-PAGE gel electrophoresis and zymography.The type of glycoprotein and glycoprotein were detected by silver nitrate chromate staining and Endo H enzyme treatment.To inactivate the recombinant xylanase Xyn A as control,and the recombinant xylanase Xyn A treated in the same Tris-HCl buffer(p H 8.0)at 42°C 72 hours laser printing paper,deinking detected.The untreated corncobs were enzymolyzed at high temperature(70~80°C)in Tris-HCl buffer(p H 8.0)and incubated for 36 hours to determine their saccharification effect.Results The codon-optimized xyn A gene was designed,synthesized and expressed in P.pastoris.The strain RX8 forming the largest hydrolysis halo in the xylan plate was used in shake flask experiments and produced an enzyme activity of 40?U/ml.After chromatography on Sephadex S-75 purified recombinant xylanase than the highest activity reached 1053 U / mg enzyme activity ratio.The size of recombinant xylanase was determined to be 75 k Da by SDS-PAGE gel electrophoresis and zymography.The results showed that the recombinant xylanase was glycoprotein,and its apparent molecular weight was significantly higher than that of its protein,indicating that the recombinant xylanase was highly glycosylated during the expression process.The optimal reaction conditions for recombinant xylanase Xyn A were 75 ° C and p H 8.0.The enzyme incubated at 100 ° C for 8h activity remains greater than 80%.Treatment of laser paper at 42°C in Tris-HCl buffer(p H 8.0)72 hours of deinking effect(247%)was higher than that of the reported Bacillus xylanase Ck Bx1D(200%).The recombinant xylanase had a saccharification efficiency of 7.44% after 36 h of untreated corn cob during high temperature enzymatic hydrolysis(70~80°C).In addition,this recombinant xylanase has better acid tolerance and stability over a wide range of p H(3.0~11.0).Conclusions Recombinant xylanase Xyn A was first expressed in Pichia pastoris,and the heat resistance of recombinant xylanase was higher than that of original xylanase.The higher activity and good thermal stability make the recombinant xylanase have great industrial application potential and economic value,and also lay a theoretical foundation for the research of other heat-resistant xylanase.