The Effect Of Gene Dosage And Culture Temperature On The Expression Of β-mannanase In Pichia Pastoris

P-mannanase(endo-1,4-D-mannanase, EC3.2.1.78) catalyzes the random hydrolysis of β-1,4-mannosidic linkages in mannan, galactomannan, glucomannan, and galactoglucomannan. It has many potential applications, for instance, pulp bleaching, coffee extract viscosity reducing, feed nutritional value improving and oil drilling. So far, β-mannanases have been extensively expressed heterologously in Pichia pastoris. The effects of gene dosage and culture temperature on the expression of Pichia pastoris were investigated for improving the production of β-mannanase in Pichia pastoris. And then through reasonably constructing Pichia pastoris with opportune gene dosage of (3-mannanase and controlling fermentation conditions to improve the expression of the exogenous protein.In this paper, we utilized the double standard curve method which were generated by qPCR with the plasmids containing GAP and GFP genes respectively, and then the copy number of heterologous gene of β-mannanase in genome of Pichia pastoris was detected by real-time fluorescent quantitative PCR. Through the qPCR,5strains harboring1,3,4,5and7copies of (3-mannanase gene were screened in order to study the effect of gene dosage on the transcription and protein expression levels of β-mannanase in Pichia pastoris. And the physiological character of strains with the different gene copy number were compared. The result showed that mRNA level increased with increasing gene copy number, but enzyme activity and the expression level of protein increased with increasing gene copy number in the beginning, and then no longer increased with the increasing gene copy number when the copy number greater than4. In addition, increasing in the gene copies of the recombinant strain gave rise to low capability of utilizing substrate, slow growth rate and reduction of final cell concentration. The exogeous gene for the all recombinant stains with different gene copy number were stably inherited in the whole100h culture process, and β-mannanase mRNA was constantly express.Through comparing the difference of transcription and protein expression levels of β-mannanase in different culture temperature. It was found that mRNA level decrease with the decreasing temperature, but exogenous protein was accumulated. And also the more significant impact of temperature on the high copy number strains was obserevd. Lowering culture temperature could evidently improve the cell aviability of the recombinant strain, thereby reducing the release of intracellular protease from the death cell, ultimately make the protease activity in the fermentation broth decrease significantly under the low temperature cultivate condition.