Evaluation of Mut+ and Muts Pichia pastoris phenotypes for expression of Fibrobacter succinogenes β-glucanase gene

According to the codon optimization of Pichia pastoris, β- glucanase gene from Fibrobacter succinogenes(Fs GLUm) was synthesized and the recombinant expression vector,named p PIC9K-Fs GLUm,was constructed. With Sal Ⅰ and Bgl Ⅱ,the p PIC9K-Fs GLUm was linearized and the β-glucanase gene was electroporated into chromosome DNA of Pichia pastoris GS115. The different methanol utilization phenotype positive strains,Mut+and Muts, were obtained after phenotype and resistance screening. In the shake flask level,the halo zones on Congo red plate produced by expression products of Mut+strain were significantly larger than that of Mutsstrain. In the fermenter level,β-glucanase activity expressed by Mut+strain was significantly higher than those of Mutsstrain in each time period. Enzyme activity,specific enzyme activity,and dry cell weight of Mut+strain reached the maximum of 6424U/m L,2607U/mg,and 123.6g/L,respectively at 96 h after methanol induction. While,that of Mutsstrain reached the maximum of 119U/m L,1867U/mg,and 113.5g/L,respectively at 108 h after induction. The above results showed that,Mut+strain of Pichia pastoris GS115 was more conducive for expression of Fibrobacter succinogenes β-glucanase gene.