Construction Of High-yield Glucose Oxidase Secreting Genetically Engineered Strain

In this experiment,the 13#is a original strain that was build in laboratory.The GOD gene in this strain has mutated by DNA shuffling technology,three amino acid sequences were mutated(D195G,T357A,L368P).In order to increase the yield of the GOD enzyme,This article focuses on three aspects exploration:In the first stage,SDS-PAGE of intracellular proteins confirm that a part of the target protein was still not secreted in the cell,and the target strain was modified from the perspective of protein expression and secretion,in transport,folding,and transcriptional regulation.The transport factor SEC56 was inserted into the pGAPZB vector,the fermentation results showed that the expression level of the target protein was not increased;three folding modifiers of CNE1,BIP and PDI were inserted into pGAPZB,the expression level of the target protein was increased in the BIP strain.The transcriptional regulatory factors HAC1 and THR were inserted into the pGAPZB,and the expression of the target protein in the strain expressing the HAC1 transcriptional regulatory factor was increased.In the second stage,the concentration of salt ions in the fermentation medium was simply optimized,When the ratio of salt ion concentration in the BSM medium was 0.5,the growth of the strain was the best,and the enzyme activity was increased from 364 U/ml to 446 U/ml.In the BIP strain constructed in the previous chapter,the enzyme activity was 530 U/ml,and the yield of the expressed was increased by 18%;the activity of the strain expressing the transcriptional regulatory factor HAC1 was 600 U/ml and the expression was increased by more than 30%.In the later work,we explored the HAC1 strain,the vgb gene derived from Vitreoscilla was expressed,and the fermentation enzyme activity was increased to 630 U/ml.In the third stage,the expression of genes from different sources was compared.GOD derived from Aspergillus niger that has been mutation,and GOX from Penicillium niche and used codon-optimized to expressed in pichia GS115.The results showed that the GOD gene,the gene was codon-optimized expression in strain that enzyme activity was 540 U/ml,and the non-codon-optimized gene was 446 U/ml;the genes from different sources was optimized in the same expression system,the activity of GOX was 640 U/ml,the GOD was 540 U/ml.The protein purification result showed that the yield of GOX protein was 1.32 g/L,while the protein yield of GOD was 1.17 g/L lower than that of GOX.