High-Lever Expression Of Thermostable Mannanase ManA In Pichia Pastoris

Mannanase is one of the main enzymes that degrade hemicellulose,and the content of hemicellulose is second next to cellulose in nature.Mannanase has been widely used in food,feed,textile,papermaking and other industries for its function.The enzyme of this study was a thermostable mannanase Man A(Accession No.KJ806637)from the thermophilic aerobic strain(caldibacillus cellulovorans)obtained upfront.Man A was secreted and expressed in Pichia pastoris to explore the impact of intracellular co-expression of HAC1 or Pichia pastoris protein folding-related molecular chaperone on Man A secretion and expression enzyme activity.Furthermore,we combined fusion polyamino acids,increased gene dosage,co-expressed of Vitreoscilla hemoglobin(VHb)and other strategies to improve the enzyme activity of secretion and expression of Man A.The study contents and results were as follows:(1)The influences of co-expression of HAC1 or protein folding related molecular chaperone on the enzyme activity of Man A secretaseThe unfolded protein response(UPR)activation factor HAC1 and 5 reported endoplasmic reticulum protein folding related chaperones ERO1? PDI? PDI1? CPR5 and Bi P in Pichia pastoris were constructed to have 6 intracellular co-expressing recombinant strain secreting Man A respectively,and the influence of the recombinant strain on the expression of Man A during the shake-flask fermentation were analyzed.The results showed that the enzyme activity of Man A in the fermentation supernatant of recombinant strains co-expressing HAC1?ERO1 and PDI increased to different extents,respectively.The recombinant strain GS115/Man A-HAC1 fermentation supernatant had the highest enzyme activity,reached 474 U/m L at 120 h,which was 26% higher than that of the control.Furthermore,selecting HAC1?ERO1?PDI to have 2 genes or 3 genes combinations,which then co-expressed in the recombinant strains secreting and expressing Man A,but the enzyme activity in the fermentation supernatant of each co-expressed recombinant strains was not further increased.(2)Construction and enzyme activity analysis of recombinant strain with high expression of Man A in Pichia pastoris.To improve the secreted enzyme activity of Man A in Pichia pastoris,four strategies were used: fusion expression of polyarginine,increase of gene dosage,intracellular co-expression of HAC1 and intracellular co-expression of hemoglobin VHb.This study found that the enzyme activity of the supernatant of the recombinant strain increased by 2%,reached 365 U/m L after fusion of polyarginine at the C-terminal of Man A.The enzyme activity of 5-copy recombinant strain was further increased by 15%,reached 391 U/m L.HAC1 was co-expressed in the cell of GS115/5Man A-R6,but the enzyme activity of the supernatant was not further improved;Then VHb was intracellular co-expressed in GS115/5Man A-R6,the enzyme activity of the supernatant of the recombinant was increased by 6%,reached 402 U/m L.However,after comparing the enzyme activities of recombinant strain GS115/Man A-HAC1?GS115/5Man A-R6? GS115/5Man A-R6-HAC1 and GS115/5Man A-R6-VHb,this study revealed that the enzyme activities in the supernatant of GS115/Man A-HAC1 were the highest,reached 474 U/m L.Therefore,co-expression of HAC1 should be given priority in the selection of strategies to improve the activity of secretion enzymes of Man A,and an effective strategy may be more advantageous than other combinations of different strategies in the expression of Man A.These results suggested that the combination of multiple strategies may not promote the secretion and expression of the exogenous protein.