The Crystal Structure And Heat-resistant Mechanism Of Thermophilic Xylanase PMxyl

As a rich renewable biological resource in nature,xylan uses biological enzymes to improve the effective use of xylan,not only to avoid environmental pollution caused by chemical reagent treatment,but also to produce products with economic value.?-1,4-endoxylanase is a kind of enzyme that acts on ?-1,4-glycosidic bond of xylan main chain.Because of its high-efficiency catalysis of xylan,it is highly concerned.Xylanase can be divided into multiple families.Among them,GH 11 family xylanase has the advantages of small molecular weight and strong specificity of substrate binding.It is widely used in practical production applications.The expression plasmid vector used in this study was obtained through preliminary laboratory reconstruction,and the amino acid sequence of xylanase rMxyl was obtained from literature review.In this study,the GH 11 family thermophilic xylanase PMxyl was developed.The physiological and biochemical characteristics of xylanase PMxyl and its three Based on this structure,we can speculate the heat-resistant mechanism of proteins.Through structural analysis and comparison,mutants S111C-N156C were designed to improve the catalytic ability and heat resistance of xylanase PMxyl.The main findings are:1.By analyzing the amino acid sequence of the xylanase rMxyl,it was found that the xylanase is composed of the GH 11 family xylanase catalytic domain and the carbohydrate binding domain.The full-length xylanase gene sequence was synthesized based on E.coli codon optimization,and on this basis,two recombinant xylanases CMxyl and PMxyl were constructed.Exogenous protein expression was carried out by BL21(GOLD)and Rosetta(DE3)expression strains,respectively.The experimental results show that the recombinant xylanase can all be expressed in soluble form,but the BL21 expression strain has more advantages in expression.After a nickel column purification,the recombinant xylanase containing the His tag and the fusion protein NusA was obtained.Enzymological test results showed that the recombinant xylanase PMxyl has a wider catalytic range and more research value.2.Purify the recombinant protein PMxyl twice with nickel column to obtain the label-free xylanase PMxyl after TEV digestion,and perform enzymological testing.The results showed that the optimal reaction temperature reached 80? and 80? t1/2 is 25 min,the optimum pH is 5.5 but it has significant activity at pH 9.0.After obtaining high purity and uniform protein solution of xylanase PMxyl,the crystallization conditions were screened.Finally,under the crystallization condition of 25%PEG 1500,crystals available for X-ray diffraction were obtained.After analyzing the diffraction data by software such as Phenix and WinCoot,the three-dimensional structure of the xylanase PMxyl was obtained with a crystal resolution of 1.40 ?.3.Recombinant xylanase PMxyl is a thermophilic xylanase,and there is a pair of disulfide bonds in its N-terminal and thumb region.By designing mutant C134A and using DTT to break the disulfide bond,the effect of two pairs of disulfide bonds on the heat resistance of xylanase PMxyl was preliminarily explored.The experimental results show that the N-terminal disulfide bond connects the first and fourth ?-sheets,making the N-terminal more stable,so it plays an important role in maintaining high activity and heat resistance.However,the disulfide bond in the thumb region has no significant effect on activity and thermal stability.In addition,by comparing with the crystal structure of 1YNA,a pair of disulfide bonds were added at the bottom of the catalytic center to construct mutants S111C-N156C,the overall enzyme activity of the mutant xylanase was improved,and the optimal temperature was increased by 5?,85?.In this paper,the recombinant xylanase PMxyl containing only the catalytic domain was constructed,and its crystal was obtained and the crystal structure was successfully resolved,which provided a theoretical basis for the subsequent research of the GH 11 family xylanase.At the same time,the role of the heat resistance of the disulfide bond in the xylanase PMxyl was revealed,and mutants with improved catalytic and heat resistance were constructed,which increased their application value in industrial production.