High Level Expression Of Recombinant Extremely Thermostable Xylanase From Pseudothermotoga Thermarum And Its Application In Beer Brewing

Xylanase is widely used in papermaking,feed,food,energy and medicine industry.In the process of industrial production,high temperature pretreatment of raw materials is a common technical method.Therefore,high temperature resistant xylanase has a very important application value.In this study,the xylanase gene from Pseudothermotoga thermarum was expressed in Escherichia coli BL21(DE3).The recombinant xylanase was purified,and applied in beer brewing.The specific research results are as follows:The recombinant vectors were constructed by pET20b and pHT43 plasmids,which were suitable for the expression of E.coli and Bacillus subtilis.The xylanase gene was successfully expressed in E.coli BL21(DE3),and the obtained recombinant protein was 130 kDa,which was consistent with the expected size of the target protein.The optimum reaction temperature of the recombinant enzyme was 95 ?,and the optimum reaction pH was 5.5.The recombinant enzyme had good thermal stability could maintain 80%activity at pH 5.0 to 8.0 for 2 h.It could still maintain 80%activity at 75 ? for 1 h.Moreover,This enzyme is strictly substrate specific and is only active against xylan.Cu²⁺,Fe³⁺and SDS could significantly inhibit the activity of recombinant enzyme,while Fe²⁺and Mn²⁺could increase the activity to 3 fold.The results of enzymatic kinetics showed that the K_m value of the enzyme was 0.36 mg/ml,the V_max was 243.9?mol/(mg·min),and the k_cat/K_m was 1455 mL/(mg·s).In the saccharization stage of beer brewing,the recombinant high temperature resistant xylan was added into the reaction system at 65 ?,and the yield of xylose and glucose was detected by liquid chromatography-differential refractive method.Without xylanase as the control,when barley was used as the raw material for beer brewing,the glucose content increased from 20 mg/ml to 37.6 mg/ml,and the xylose increased from 0.017 mg/ml to 1.242 mg/ml.When wheat was used as raw material for saccharification,the glucose content increased from 21.9 mg/ml to 31.8 mg/mL and the xylose increased from 0.003 mg/ml to 0.690 mg/ml.The recombinant xylanase obtained in this study has good high temperature resistance and acid resistance,and has good catalytic efficiency.Therefore,it has important application value and can provide important theoretical basis and practical significance for high temperature hydrolysis process.