Gene Mining,heterologous Expression And Rational Engineering Of Cold-active And Alkaline β-1,4-endoglucanase

β-1,4-endoglucanase（EC3.2.1.4）can randomly cutβ-1,4-glycosidic bonds,which is widely used in laundry,textile,paper and renewable energy industries.In the laundry industry,the application ofβ-1,4-endoglucanase can improve the softness of cotton fibers and increase the washing efficiency.At present,local detergent enzymes have not been effectively developed,it is important to obtain newβ-1,4-endoglucanases for the development of domestic detergent enzyme.In this thesis,three novel cold-adaptedβ-1,4-endoglucanase genes were obtained through gene mining from NCBI website,expressed in Escherichia coli.Moreover,the alkaline tolerance ofβ-1,4-endoglucanase was improved through rational design methods such as sequence matching and protein p K_a value calculation design.Finally,the mechanism of the improved alkaline tolerance of endoglucanase was preliminarily analyzed.The main findings of this thesis are as follows.（1）Three novelβ-1,4-endoglucanase genes were screened from the database by gene mining and they were efficiently expressed in E.coli BL21（DE3）through vector p ET-22b（+）.After analyzing their catalytic activity and enzymatic properties,Eg DC could maintain more than 75%relative activity after treatment at 20°C for 1 h,indicating its potentially value for applications in laundary industry.（2）Two strategies,amino acid sequence comparison and calculation of p K_a values of amino acid residues,were used to identify 28 potential sites related to alkaline resistance.Thirty-six single mutants were constructed by site-directed mutagenesis.The specific enzyme activities and enzymatic properties were characterized,five single mutants,V73K,N122R,P131K,A168K and A208K,with improved alkali-tolerance were obtained by screening.Among them,the mutant V73K had improved alkaline tolerance and increased catalytic performance(k_cat/K_m value)by 57.1%,and the mutant P131K had the highest increase in specific enzyme activity,with 21.8%increase compared with the wild type.（3）A double mutant V73K/A168K with valuable detergent application was obtained.Five combined mutants V73K/N122R,V73K/P131K,V73K/A168K,V73K/A208K and V73K/N122R/A168K were constructed.The optimum p H of combined mutant V73K/A168K raised to p H 7.5,with 77.1%and48.5%relative enzyme activities remaining after one-hour treatment at p H8.0 and p H9.0.Meanwhile,the mutant k_cat/K_m value reached 1229.71 s^-1·mg^-1,and the catalytic performance was improved by136.34%compared to the wild type.（4）The p K_a values of key acid/base catalytic residues such as GLU¹⁵⁵ and GLU²⁵⁹,respectively,were heavily relevant to the optimal reaction p H of enzyme.This influence was mainly due to the fact that the ionization state of the key amino acids would interact electrostatically with other charged residues,thus changing the internal charge state of the protein.Further internal charged-force analysis and surface potential comparison also revealed that the introduced basic amino acids contribute to the stability ofβ-1,4-endoglucanase in an alkaline environment.