Cloning,Heterologous Expression And Enzymatic Properties Of Alkalitolerant Xylanase From Cellulomonas Bogoriensis

Cellulose,hemicellulose and lignin are the main constituents of plant cell walls.However,the hemicellulose composed of xylan is the second most abundant polysaccharides after cellulose,and it is also an important renewable resource in nature.The xylanase is indispensable for efficient usage of xylan,and it has a wide range of application in feed,food,paper,energy conversion and so on.In recent years,with the continuous development of enzymatic papermaking technology and the increasing demand for the characteristics of the enzyme itself in the production process,enzymes with special properties,such as alkali-resistant xylanase,have become hot spots in the field of biotechnology development.In this paper,Cellulomonas bogoriensis was used as the research object,the conditions for the production of xylanase from this strain were optimized,and the genomic information of the strain was used to clone and express the five xylanase genes,and the enzymatic properties of the recombinant enzyme were performed.The main findings are as follows:1?By single factor experiments to determine the optimal conditions for the production of xylanase from the starting strain Cellulomonas bogoriensis:cultivation time 4 d,medium liquid volume 100 mL/500 mL,cultivation temperature 30?,medium initial pH 10.5,the optimum carbon and nitrogen source are bran and yeast extract,content 1%?w/v?,NaCl concentration 3%?v/v?,inoculation content 4.0%?v/v?.The determination of xylanase crude enzyme showed optimum reaction temperature is 60?with good temperature stability in the range of 4-40?;optimum pH is 7.0 with good pH stability in the range of pH 5-12.2?By analyzing the genomic information of Cellulomonas bogoriensis,five endo-xylanase genes derived from this strain were cloned and expressed.Determine the optimal induction conditions for the five recombinant proteins.The best induction temperature for Xyn370?Xyn425?Xyn486 is 18?,and Xyn393?Xyn466 is 30?,The optimal IPTG final concentration is 0.075 mM.Using affinity chromatography to obtain the pure enzyme components of the five recombinases,molecular sizes of 41.6kDa?43.9 kDa?47.3 kDa?50.5 kDa and 51.7 kDa respectively,specific activity was86.4 U/mg?88.2 U/mg?79.3 U/mg?80.3 U/mg and 89.3 U/mg respectively.3?Five recombinase optimum temperature was 60??50??40??40??60?,respectively.Both of these five enzymes have good thermal stability of 4-70?,explain that these recombinant xylanases have a certain degree of heat resistance.The optimum pH are 7.0?8.0?8.0?8.0?9.0,respectively,in the range of pH 5.0-11.0 the remaining activity of the xylanase is more than 80%,indicating that the recombinant enzyme are alkalitolerant xylanase.4?Five recombinant xylanases show good tolerance to some metal ions and have very good salt tolerance.The five kinds of recombinases has broad substrate specificity,and the enzyme activity of the soluble beech xylan is the highest.When using woody xylan as a substrate,K_m is 0.65 mg/mL?0.40 mg/mL?0.30 mg/mL?1.13mg/mL and 0.74 mg/mL respectively,V_max is 48.92?mol/min/mg?65.92?mol/min/mg?2.68?mol/min/mg?94.47?mol/min/mg and 59.52?mol/min/mg,respectively.In this paper,expressed and purified of the five recombinases have excellent characteristics of high temperature resistance and saline-alkali resistance,and are well tolerant to certain metal ions and chemical reagents.They provides materials for studying the catalytic tolerance mechanism of xylanase,and also provides a new enzyme source for papermaking,washing,resource development and utilization,and environmental protection.