Cloning,Recombinant Expression And Molecular Modification Of Psychophilic Xylanolytic Enzymes

Xylan is one of the major components of plant cell wall,a large variety of cooperatively acting enzymes are required for complete hydrolysis of xylan because of its complex structure. Xylan degrading enzymes have been widely used in feedstuffs, food, paper and bioenergy industries. In recent years, it has been found that the application of psychrotestine degrading enzymes in food processing and other industries can effectively reduce the loss of active ingredients, flavor substances and nutrients, while reducing production costs. However, the vast majority of xylanases and xylosins currently found belong to the high temperature enzyme (http://www.brenda-enzymes.org), which has been reported to have a low sequence of gene sequences and enzymatic properties Xylosidase is still lacking, it is urgent to explore the excellent nature of the cold xylanase and xylosidase gene resources, and its psychological mechanism of the study, the study obtained a sequence and the nature of the novel cold xylanase Xyn27, And the psychophysical mechanism of the cold xylosidase AX543 was analyzed and studied.The specific contents are shown as follows:A novel xylanase of family 10 (Xyn27) was obtained from the Acremonium by Touch-down PCR and thermal asymmetric interlaced (TAIL) PCR methods. Xyn27 contained a putative signal peptide (20 residues) and a catalytic motif of GH10 (346 residues) and shared the highest identity (83%) with the reported GH10 xylanase(XM 003662144). The recombinant Xyn27 was successfully expressed in Pichia pastoris GS115 and the optimal induction condition was 35? for 48 h. Xyn27 was a cold active xylanase, which shows its highest activity at 35? and still had 60.25%, 38.70% and 10.8%relative activity at 20?, 10? and 0?. Further analysis showed that Xyn27 has fewer arginine and more alanine residues compared with its mesophilic or thermophilic counterparts. The optimal pH of Xyn27 was 7.0, and it was stable after incubating under the pH range from 3.0 to 9.0 for 1 h. Besides, Xyn27 exhibited superior metal ions tolerance than other GH10 cold active xylanases, which is tolerant to most metal ions and organic solvents, and significantly enhanced by heavy metal ion Ca2+, Mn2+ and Zn2+. In addition,the Km, Vmax, kcat, and kcat · Km-1 against beechwood xylan were 13.42 mg · mL-1, 9.07 umol· min-1 · mg-1, 192.98 min-1 and 14.38 mL · min-1 · mg-1, and Xyn27 could completely degraded xylan into xylobiose. The features of cold activity and metal ion tolerance suggested the potential application of xylanase Xyn27 in basic research and various industries like food possessing.The optimal temperature of xylanase AX543 is 25 ?, which is the lowest temperature enzyme reported at present. It is concluded that the difference of loop structure may be the main reason, through a series of sequence analysis and structural comparison, loopI (31-46),loop2 (54-59), loop3 (164-183), Loop4 (209-224) and loop5 (296-298). For these sites, the primers were designed to mutate successfully. Six plasmids were successfully constructed.The first five mutants were successfully expressed in Pichia pastoris, but because of the lower activity, the next step was not studied.