Gene Clonging,Expression And Molecular Improvement Of Novel Glucose Oxidase

Glucose oxidase?GOX?is widely used.Few new types of GOX have been reported other than those derived from Aspergillus and Penicillium.In this study,glucose oxidase producing strains were screened from fungi preserved in our laboratory by method of plate coloration.The results showed that only psychrophilic Cladosporium neopsychrotolerans SL16 showed GOX activity.According to its genome sequencing data,the CngoxA encoding GOX was cloned from C.neopsychrotolerans SL16.In addition,according to the sequence similarity of GOX,five possible GOX coding genes were cloned from C.tianshanense SL19 and Aspergillus niger MJ5,named CngoxB,AngoxA5,AngoxA7,AngoxA8and AngoxA3.The proteins encoded by CngoxA and CngoxB shared the highest identity of 35%with the experimentally verified GOXs,and the identity between them was 80%,both of them belong to new type GOXs.The protein encoded by AngoxA5 shared the highest identity of 97%with the experimentally verified GOX,while AngoxA7,AngoxA8 and AngoxA3 were about 50-68%.Six genes were expressed in Pichia pastoris,only CngoxA and AngoxA5 were successfully expressed.The CnGOXA from C.neopsychrotolerans SL16 showed highest enzyme activity at 20?and pH 7.0,and it belongs to psychrophilic enzyme,had above 75%relative activity at 0?.It was stable at pH 6.0–9.0,with a half-life at 40?for 15 min.The specific activity of CnGOXA was 90U·mg^-1,K_m and V_max were 103 mM and 90.1?mol·min^-1·mg^-1,respectively.CnGOXA had strong resistance to SDS,and 5 mM and 10 mM SDS had no significant effect on its activity.The properties of the AnGOXA5 was similar to those experimentally verified GOX from A.niger.The activity could not be detected significantly when CngoxB gene from C.tianshanense SL19 was expressed in P.pastoris.After sequence and structure analysis,we found that the C-terminal of CnGOXB was shorter than CnGOXA.We obtained CngoxB-M1 by splicing the corresponding nucleic acid sequence of CngoxA into CngoxB.CnGOXB-M1 was expressed successfully in P.pastoris,and its properties were similar with the CnGOXA.But the specific activity of CnGOXB-M1?123.8 U·mg_?^-1higher than CnGOXA.In order to improve the stability and catalytic activity of CnGOXA,we studied the mutations of CnGOXA.The mutant CnGOXA-M1 was constructed by introducing a disulfide bond?Y169C-A211C?at Y169 and A211 sites of CnGOXA.The optimum pH of CnGOXA-M1 expressed in P.pastoris was the same as that of wild CnGOXA,and the optimum temperature was 25?,which was 5?higher than that of CnGOXA.Thermostability and pH stability were greatly improved.The half-life at 40°C was 48 times higher than that of wild type?720 min vs.15 min?.The range of pH stability was extended from pH 6.0-9.0 to pH 3.0-12.0,with the specific activity was 123.8 U·mg^-1,which was 37%higher than that of CnGOXA.The mutants were constructed by introducing pi-pi bonds,interaction-network of hydrogen bonds,salt bridges and surface charge interaction based on CnGOXA-M1,which named T525H,G147R,E74K,E150K and E476K.The thermal stability of the above mutants expressed in P.pastoris were higher than that of CnGOXA-M1.The mutant CnGOXA-M2 was obtained by combining above five mutations with CnGOXA-M1.Compared with CnGOXA-M1,the remaining activity of CnGOXA-M2 was increased by about 200-folds?79.8%vs.0.4%?at 50?for 1 h and the specific activity was 166.2 U·mg^-1,which was increased by 34%.CnGOXA-M1 was applied to bread baking test.The results showed that the optimum amount of CnGOXA-M1 and GOX from A.niger added in bread were 6 and 4 U·kg^-1,and the volume to mass ratios were 6.4 and 6.2 cm³·g^-1,respectively.Compared with the control group without enzymes(5.9 cm³·g^-1),CnGOXA-M1 and GOX from A.niger could effectively promote the volume of bread and CnGOXA-M1 showed better.In this study,six glucose oxidase genes were cloned.Three of them were successfully expressed in P.pastoris and GOX activity was detected.The mutation of CnGOXA from C.neopsychrotolerans SL16 was studied.The thermal stability,pH stability and catalytic activity of CnGOXA were effectively improved.CnGOXA-M1 could effectively promote the increase of bread volume in bread baking experiment.In addition,the CnGOXA-M1 and CnGOXA-M2 kept high activity under low temperature,neutral-alkaline condition and strong resistance to the SDS,which makes them have potential application in the washing detergent and aquatic feed additive industry.