Heterologous Expression And Molecular Modification Of Xylanase From Clostridium Clariflavum

Xylanase is a kind of important industrial enzyme.In order to develop xylanase suitable for various applications,the researchers are being looking for new xylanase sources as well as molecular modification of existing xylanase.In this study,the multi-domain xylanase gene Clocl-2441 from C.clariflavum was reconstructed with different domains.The optimal recombinant xylanase rXyn2441GH10 was obtained and its enzymatic properties were determined.The molecular modification of rXyn2441GH10 was carried out by the truncated expression to further improve the enzymatic properties.Finally,site-directed mutagenesis was used to determine the catalytic key amino acids of the enzyme.The major results are as follows:?1?Recombinant expression of different domains and determination of enzymatic properties.According to the domain information of Clocl-2441 analyzed by NCBI,6recombinant strains were constructed by combination of different domains.The activity of the recombinantenzymesr Xyn2441GH11,rXyn2441GH11C,rXyn2441GH11CD,rXyn2441GH10 rXyn2441GH10D and rXyn2441GH10DC were 1.87,2.07,1.25,6.04,2.13and 1.89 fold of that of the full-length enzyme,respectively.The rXyn2441 GH10 showed the optimum temperature and pH of 70°C and 7.0,respectively,belonging to the neutral thermophilic xylanase.rXyn2441GH10 was stable at temperature below 65°C and pH between4.0 to 9.0.5 mM of Mg²⁺in all metal ions tested increases the enzyme activity by 79.2%.The kinetic parameters of recombinant enzyme to wards beech xylan substrate were V_maxax 1691.5U·mg^-1,K_m 2.5 mg·m L^-1,k_cat 1236.4 s^-1,k_cat/K_m 494.6 m L·mg^-1·s^-1.The main products of rXyn2441GH10 hydrolysis are mainly xylobiose and xylotriose.?2?Molecular modification of xylanase rXyn2441GH10.Through multi-sequence analysis of the GH10 family xylanase,it was found that the N-terminus of rXyn2441GH10 has very flexible and disordered residues,and an N-terminal truncated mutant were expressed in a design experiment.The enzymatic activities of the five mutants rXyn2441GH10N-10,rXyn2441GH10N-20,rXyn2441GH10N-31,rXyn2441GH10N-37,rXyn2441GH10N-41 were1.04,1.79,1.74,0.29,0.16-fold that of rXyn2441GH10,respectively.The rXyn2441GH10N-10,rXyn2441GH10N-20,and rXyn2441GH10N-31 were isolated and purified for their enzymatic properties.Compared with rXyn2441GH10,the optimum temperatures of rXyn2441GH10N-10 and r Xyn2441GH10N-20 did not change but the temperature stability increased.The optimum temperature of rXyn2441GH10N-31 decreased by 5°C while the thermal stability decreased.The maximum reaction rates of rXyn2441GH10N-10 and rXyn2441GH10N-20 increased by 10.8%and 15.6%,respectively,compared to rXyn2441GH10,while r Xyn2441GH10N-31 decreased by 6.2%.?3?Analysis of conservatively catalyzed key residues in rXyn2441 GH10.The conserved amino acids were selected by homologous sequence alignment and homology modeling simulation,and the selected amino acids were replaced with Ala by site-directed mutagenesis.Seven catalyzed amino acid residues were identified:His111,Glu160,Gln238,His240,Glu271,Trp312 and Trp320.Among them,Glu271 acts as a nucleophile and Glu160 acts as an acid catalyst for proton donors.His111,Gln238,His240,Trp312,and Trp320 form a hydrogen bonding network around the two active residues and are responsible for binding to the substrate and stabilizing the conformation of the active residues.