| Chitin is widely found in nature and has functions such as inhibiting cancer,eliminating bacteria,improving diabetes,and protecting bones.However,because it is insoluble in water and application in vitro is greatly limited,while chitin hydrolysates N-acetylchitooligosaccharides,GlcNAc,and GlcN are soluble in water and have same functions such as antibacterial and cancer suppression.At present,the main method for degrading chitin to produce the above functional products is chemical method,but there are problems such as complicated production steps,low yield,and serious pollution.By comparison,Chitinase catalysis has the advantages of environmental protection,no pollution,and simple and convenient operation.However,there are some problems about chitinase such as low hydrolysis efficiency and non-single product.Therefore,it is necessary to discover new chitinase to solve such problems.GlcN is widely used in daily life,but chemicals are used for industrial production to produce GlcN,not only many by-products,but also the GlcN produced in the form of hydrochloride,which is limited in the field of medicine.The direct hydrolysis of shrimp shell powder by the whole enzymatic method to produce GlcN can theoretically ensure the purity of the product and better active form.At present,there has been no report on the production of GlcN by the enzymatic method,so it is great significance to develop a full-enzyme method for the production of GlcN.In this study,using bioinformatics technology to find a gene that may have chitinase activity from Myxococcus xanthus,named Mx2882.The gene is recombinantly expressed in prokaryotes by genetic engineering technology,and the obtained recombinant enzyme has the function of hydrolyzing chitin.Biochemical characteristics and substrate specificity were studied by microplate reader technique.The evolutionary tree analysis,protein three-dimensional structure simulation map and multiple sequence alignment were used to infer that the recombinant chitinase belongs to the GH18 family,and the three active sites Asp323,Asp325 and Glu327 in the catalytic region DXDXE sequence of the enzyme were subjected to site-directed mutagenesis and test mutants' activity.On this basis,the GlcN was produced by a multi-enzyme method and the reaction conditions were optimized.Finally,the enzymes in the multi-enzyme method were immobilized.The specific research contents and results are as follows:1.Gene cloning,expression and activity testing of chitinaseA gene encoding a chitinase that is identical in size to the target gene(1731 bp)was cloned from Myxococcus xanthus.Expressed in E.coli by recombinant technique,and the chitinase was purified by a nickel affinity chromatography column.The protein molecular weight of chitinase was about 60 KDa by denaturing polyacrylamide gel electrophoresis(SDS-PAGE),which was consistent with the theoretical value of 61KDa,and named Mx2882.The colloidal chitin was used as a substrate to react with the chitinase crude enzyme solution,and a target band consistent with the standard sample(chitobiose)was found by thin layer chromatography.It was found that the enzyme hydrolyzed colloidal chitin and produced chitobiose.The chitinase purified by nickel column was reacted with colloidal chitin and tested by UPLC to further verify that the product was chitobiose,ensures the purity of the product.2.Study on the biochemical characteristics of chitinase,specificity studies and enzyme catalytic site activityUsing colloidal chitin as the reaction substrate,the product chitobiose was converted to GlcN by hexosaminidase Sn384 and deacetylase CmCBDA,and the amino group in GlcN was detected by microplate reader to study chitinase.Biochemical properties of Mx2882,The results showed that the optimal reaction temperature of chitinase Mx2882 was 55?,but the enzyme maintained high activity in the temperature range of 37? to 55?.Although the optimum pH is 5,the enzyme activity at pH 8 is also maintained at 80%or more.The metal ion had no effect on the activity of chitinase Mx2882.The enzyme was found to have an optimum temperature of 55? but the enzyme was easy to lose activity at this temperature,and the enzyme passed the 72 h at 37? and the activity remains above 80%.This enzyme can hydrolyze chitin,but can not hydrolyze chitosan,so it is specific for N-acetylation of the substrate.Through evolutionary tree analysis and three-dimensional structural simulation of the protein,it was found that the chitinase Mx2882 belongs to the GH18 family and site-directed mutagensis was performed to three catalytically active sites Asp323,Asp325 and Glu327 in the catalytic region DXDXE sequence of recombinant chitinase Mx2882.The three mutants of the enzyme were found to be inactive by the microplate reader and Congo red dying,which proved that any one of the catalytic active sites of the enzyme was changed,and the enzyme will lose the activity.3.One-pot multi-enzyme production of glucosamineChitinase Mx2882,hexosaminidase Sn384 and deacetylase CmCBDA successfully hydrolyzed colloidal chitin and shrimp shell powder to produce G1cN in a multi-enzyme experiment.The optimal dosage ratio of enzyme was 1:3:15(chitinase Mx2882:hexosaminidase Sn384:deacetylase CmCBDA)by single factor experiment.The optimum pH was 8,and the optimal reaction temperature was 37?.Based on this,orthogonal experiments were carried out to find the optimal reaction combination of one-pot multi-enzyme method,1:3:16.6(chitinase Mx2882:hexosaminidase Sn384:deacetylase CmCBDA),pH 8 and 37?.Although this condition is not the optimal action condition of chitinase Mx2882,it can maintain high activity.The optimum hydrolysis efficiency was obtained under the conditions,at which time 40 mg of shrimp shell powder was hydrolyzed to obtain 2.48±0.08 mg of GlcN.Among the three factors,temperature has the greatest influence on the reaction,followed by pH,and finally the ratio of enzyme dosage by analysis of variance.The above three enzymes were immobilized on chitin powder.And in this condition,40 mg of shrimp shell powder was hydrolyzed to obtain 5.78±0.01 mg of GlcN. |
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