High-level Expression Of A Highly Thermostable ?-mannanase By Pichia Pastoris And Its Mechanism Analysis Of Thermostability

?-Mannanase?EC 3.2.1.78?can randomly catalyze?-1,4-mannosidic linkages in the main structure of mannans by releasing mannose and mannan-oligosaccharides.As an important industrial enzyme,?-mannanase is widely used in animal feed,food processing,biorefining,textiles and second generation biofuels production.However,high temperature treatment is usually involved in some applied fields,such as granulation process of animal feed and bioenergy production,which requires the developedb-mannanases possess excellent heat resistance.At present,theb-mannanases in the market are often below the requirement for high thermostability.Therefore,it's urgent to develop a highly thermostable?-mannanase,which has a good application prospect.In this study,a thermophilic Bacillus subtilis TBS2,which could produceb-mannanase,was isolated and screened from a hot spring?water temperature>68??.Using TBS2 as the original strain,we obtained a recombinant highly thermostableb-mannanase?ReTMan26?expressed by P.pastoris through gene coloning.After purification,the enzymatic properties of ReTMan26 were determined and its mechanism of high thermostability was analyzed.On this basis,the expression level of ReTMan26 in P.pastoris was greatly improved through codon optimization.In addition,we determined that crude glycerol,a byproduct derived from biodiesel,could be used as a cheap carbon source for the ReTMan26 production.The main findings are listed as follows:?1?Through morphology,physiological,biochemical as well as cultural characteristics,16S rDNA sequence alignment and construction of phylogenetic tree,theb-mannanase producing bacterium,which was isolated from a hot spring,was identified as a thermophilic Bacillus subtilis?TBS2?.The TBS2 could grow at pH range from 4.0 to 9.0 and in temperature range from 35? to 80? with the optimum pH at 6.0-7.0 and optimum temperature at 55?,respectively.Moreover,the optimum pH of theb-mannanase?BS-Man?produced by TBS2 was 6.0,and the optimum temperature was 55?.After treatment at 90? in aqueous solution for 10 minutes,the residual enzymatic activity of BS-Man reached 63.6%.?2?According to the conserved sequences ofb-mannanases produced by other Bacillus bacillus strains from NCBI,we designed primers and successfully cloned a highly thermostableb-mannanase gene TBS2-Man from the thermophilic Bacillus subtilis TBS2.TBS2-Man was 957 bp in length,which encoded 318 amino acids.Theb-mannanase belongs to the glycoside hydrolase 26 family through the analysis of protein structure and gene sequence,and contains three putative N-glycosylation sites?N8,N26 and N255?and two cysteines?C48 and C68?.Based on the above,a P.pastoris strain?Orig-pPICZ?A-X33?was constructed and screened to express the recombinant highly thermostableb-mannanase?ReTMan26?,and the ReTMan26 expression levels by P.pastoris Orig-pPICZ?A-X33 were312 U×mL^-1 in shake-flask cultivation and 5435 U×m L^-1 in 50 L bioreactor high-cell-density fermentation,respectively.?3?After purification of the ReTMan26 produced by Orig-pPICZ?A-X33,the enzymatic properties of ReTMan26 were investigated.ReTMan26 was stable at a range of pH from 2.0to 8.0 with optimum pH 6.0,and ReTMan26 was active at broad range from 20? to 100? with optimum temperature 60?,respectively.The ReTMan26 retained 58.6%of its maximum activity after treatment at 100? in aqueous solution for 10 min.The activity of ReTMan26 was stimulated by Mg²⁺and Mn²⁺,but inhibited by Ag⁺,Hg²⁺and SDS at concentration of 10 mmol×L^-1.The Michaelis constant?K_m?and the maximum velocity(V_max)values of ReTMan26 were 4.35 mg×m L^-1 and 1612 U×mg^-1 protein,respectively.Moreover,the ReTMan26 showed strong resistance to digestive proteases?pepsin and trypsin?.All of the above properties suggested that,in addition to other potential industrial applications,ReTMan26 was suitable to be used as a feed enzyme.?4?In order to investigate the high thermostability mechanism of ReTMan26,amino acid sequence alignment and protein three-dimensional structure analysis were performed.The results showed that shorter protein sequence,some different animo acids,disulfur bond and N-glycosylation might be the basic reasons for the high thermostability of ReTMan26.To further investigate the effects of N-glycosylation and disulfur bond on the stability,especially on thermostability of ReTMan26,the N-glycan was removed by Native Protein Deglycosylation Kit,and two cysteines?C48 and C68?were eliminated using site-directed mutagenesis,respectively.The results showed that N-glycosylation could increase the optimum reaction temperature and thermostability of ReTMan26 about 5? and 7?,respectively.Furthermore,N-glycosylation could increase the resistance of ReTMan26 to pepsin and trypsin by 23.7%and 25.6%,respectively.However,the disulfur bond had no effect on the optimum reaction temperature and improving the resistance of ReTMan26 to the above two digestive proteases,but it could increase thermostability about 3?.?5?To further increase the expression level of ReTMan26 by P.pastoris X33,the original gene of ReTMan26 TBS2-Man?Orig?from TBS2 was subjected to codon optimization,and the recombinant P.pastoris CoOp-pPICZ?A-X33 containing the codon-optimized gene?CoOp?was constructed as a new producing strain.Under identical conditions,the expression level of ReTMan26 by P.pastoris CoOp-pPICZ?A-X33 was 10750 U×mL^-1 in high-cell-density fermentation,which was increased by 98.6%than that by the original recombinant P.pastoris Orig-pPICZ?A-X33(5412 U×mL^-1).Furthermore,a Kozak sequence GCCACCATG was adapted onto the initiation codon ATG of?-factor,which is the signal peptide of ReTMan26,located in front of CoOp.However,the result indicated that Kozak sequence adaptation had no effect on increasing the expression level of ReTMan26 in P.pastoris,but it has a certain reference function for construction of recombinant P.pastoris expression system in the future.?6?In order to reduce the production costs of ReTMan26 and make full use of the waste resources,the crude glycerol,a main byproduct derived from biodiesel production,was used as the sole carbon source during the rapid growth of P.pastoris.The results showed that the soap in crude glycerol at the concentration below 0.3%?w/v of medium?would not inhibit cell growth and ReTMan26 production in high-cell-density fermentation,and non-pretreated crude glycerol could replace pure glycerol for the production of ReTMan26 under this premise.Therefore,the total production costs of ReTMan26 using non-pretreated crude glycerol were⁴.2%lower than those using pure glycerol.