High-Level Expression Of Thermophilic Enzyme And Enhanced Enzyme PH Adaptation Based On Statistical Analysis Of Sequences

Thermophilic enzyme has high activity, excellent thermostability and resistance to organic solvent, which makes it good choice for many industrial applications. However, the biomass yield and the expression level are remarkably low, and extreme pH environment makes the enzymes lose activity quickly. These limit wider application of thermophilic enzymes. Heterogenous high-level production in a proper host and developing a novel strategy to enhanced enzyme p H adaptation have great potential to solve those problems. In this study, we achieved the high-level heterogenous expression of thermophilic esterase and xylanase, and enhanced enzyme pH adaptation using a new method based on statistical analysis of GH11 family sequences.In order to establish the high-level expression system, we constructed the highlevel expression system of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in P. pastoris and E.coli. The gene encoding EST2 was cloned to pET28a(+) and pPIC9 K and transformed to BL21(DE3)-codon plus(RIL) and GS115. The recombinant EST2 expressed in E.coli and P. pastoris showed very similar properties. The result of high cell-density fermentation revealed that the recombinant EST2 was predominantly expressed extracellularly with activity of about 960 U/m L in P. pastoris and the EST2 activity increased to 13700 U/mL in E.coli. After further optimizing fermentation conditions, we found the cell density starting induction effected the expression level. When we started induction using IPTG at OD600 = 120, the final OD600 reached 172, and the dry cell weight, activity and yeild were respectivly 60.8 g/L, 14800 U/mL and 3.2 g/L. The yield was higher than most of other studies. We constructed the heterogenous high-level expression system of thermophilic xylanase CbX-CD from Caldicellulosiruptor bescii DSM 6725 in E.coli and P. pastoris, and optimized the conditions of high-density fermentation. CbX-CD expressed in E.coli had higher optima pH value and specific activity. After further optimizing fermentation conditions, we found the cell density induction was important. When we started induction at OD600 = 90, the final dry cell weight reached 70 g/L, the activity was 45000 U/mL and the yield per hour was 2140 U/mL/h. This yield was higher than most of data reported, which provided CbX-CD wider applications in bioindustry.The complex protein microenvironment and amino acid interactions have multiple effects on the dissociation state of catalytic residues. The traditional methods such as rational design cannot exactly locate the key sites related to pH Optimum, so exploring an efficient strategy shifting pH optimum of enzyme has important significance. GH11 family xylanses have a wider pH optimum range and good research foundation, which makes it be a good model for optima pH study. After analysis of protein domain and identity, we selected 115 xylanases to annotate with pH optimum from more than 1000 sequences, and constructed a new library. Then we used p I value and hydrophobic index to descript the 115 sequences. The result of artificial neural networks and Lasso linear regression analysis demonstrated that there were eight potential sites related to pH optimum. According to the result of mutagenesis in CbX-CD, the pH optimum of F54 W,S55D,A165 E,D175Y and Q176 E shifted 0.5-0.75 units towards acid area, meanwhile kept more than 80% activity. Through analyzing the 3D structure models, we found the electrostatic interaction affected the pKa of catalytic residue Glu92, and then shifted the optima pH value of CbX-CD. This study explored a new strategy based on statistical analysis of the protein sequences which shifted the optima pH meanwhile kept the activity.