Overexpression,Characterization And Thermophilicity Of Xylanase B From Thermotoga Neapolitana (DSM 4359)

Xylan is an important class of lignocellulosic biomass resources.Xylanase is a key enzyme in the xylan degrading enzyme system and plays an important role in biomass conversion.At the same time,xylanase also has a wide range of applications in industry,such as feed industry,food industry,paper and pulp industry.Because of its low yield and poor thermal stability,natural xylanase cannot be mass produced in industry.Developing a novel xylanase from thermophilic bacterial and improving the thermal stability of xylanase by protein engineering are effective ways to reduce the cost of xylanase industrial applications.Thermotoga neapolitana is a hyperthermophile that produces a variety of thermostable hydrolytic enzymes.In this paper,the mature peptide sequence coding for xylanase B was cloned from Thermotoga neapolitana;the xylanase B gene was highly expressed by using genetic engineering methods,and to explore the enzymatic activity of the xylanase B.the thermostability of xylanase B was improved by the method of protein semi-rational design.The main contents of this study are as follows:?1?Construction of recombinant overexpression plasmids for xylanase B from Thermotoga neapolitana?DSM 4359?.The genome of Thermotoga neapolitana was used as a template,the mature peptide sequence coding for xylanase B was amplified by PCR;and the recombinant plasmid pHsh-xlnB was constructed by using pHsh-T vector which is a novel heat shock expression vector;recombinant xylanas was expressed in E.coli clearly.?2?Purification and characterization of recombinant xylanase.The recombinant xylanase was purified by heat treatment and nickel affinity chromatography.The optimum temperature of Tne-xlnB was 75°C,the optimum pH was 6.0,and it had good thermalstability;the K_m and V_max of Tne-xlnB towards birchwood xylan were1.086 mg/mL and 191.76 U/mg;1 mM Mg²⁺,Co²⁺,Sr³⁺,Ba²⁺,Ca²⁺promoted the enzymatic reaction of Tne-xlnB,1 mM Zn²⁺,Cd²⁺,Fe³⁺,Cu²⁺,Ni²⁺,Cr³⁺had a strong inhibitory effect on the enzymatic reaction of Tne-xlnB,1 mM Fe²⁺and Mn²⁺had a weak inhibitory effect on the catalytic reaction of Tne-xlnB,1 mM K⁺,Na⁺,NH₄⁺,EDTA had little effect on the catalytic activity of Tne-xlnB.?3?Thermostability improvement of Tne-xlnB.The amino acid sequence homology of Tne-xlnB was analyzed and the protein structure of the xylanase was simulated to identify the mutation site;Site-directed mutagenesis of the amino acids at the?10 helix,?11 helix,and?12 helix of Tne-xlnB;pHsh-xlnB was used as a template and the mutant Tne-xlnB-?10/11/12 was successfully constructed using the inverse PCR technique;the successfully constructed mutant expression plasmid was transformed into E.coli.the mutant Tne-xlnB-?10/11/12 was purified by heat treatmentand nickel affinity chromatography?The optimal reaction temperatures of Tne-xlnB-?11/12 and Tne-xlnB-?10/11/12 were 80°C and 85°C;K_m of Tne-xlnB-?10/11/12 towards birchwood xylan was 0.2943 mg/mL and V_max was376.42 U/mg;The thermostability of Tne-xlnB-?10/11/12 showed that activity was maintained at above 77%after incubating at 90°C for 1 h,but The thermostability of Tne-xlnB showed that activity was maintained at above 55%after incubating at 90°C for 1 h;The thermostability of Tne-xlnB-?11/12 showed that activity was maintained at above 45.21%after incubating at 90°C for 2.5 h,but The thermostability of Tne-xlnB showed that activity was maintained at above 38.72%after incubating at90°C for 2.5 h.The thermostability of Tne-xlnB-?11/12 and Tne-xlnB-?10/11/12 was significantly higher than that of Tne-xlnB.