Structural Analysis Of Dextranase From Penicillium Cyclopium CICC-4022 And Study On Its Molecular Weight Regulation And Degradation

Dextranase（dextranase;E.C.3.2.1.11）can specifically degrade theα-1,6 glycosidic bond in dextran,which is an important biological enzyme material widely used in food and medicine fields.Dextran enzymatic method has the advantages of environmental protection,simple operation and high specificity,which can solve the pollution of large molecular weight dextran and improve the quality of sucrose in the production process.In the field of food and medicine,it can be used to controllably produce dextran with specific molecular weight,which is one of the effective means to optimize and upgrade the whole industrial chain of sucrose.In the medical field,it can be used to prevent and treat dental caries.Penicillium cyclopium CICC-4022,which produces dextranase（PC-Edex）,was screened by our group in the early stage,and the enzyme production conditions were further optimized in this paper.The structure and active site of PC-Edex produced by P.cyclopyum CICC-4022 were further studied,and the interaction mechanism between PC-Edex and dextran molecules was explored,which provided theory and practice for analyzing the process mechanism of enzyme degradation of dextran and obtaining effective technical means of dextran with a certain molecular weight by enzymatic hydrolysis regulation.The main contents are as follows:By optimizing the fermentation conditions of P.cyclopium CICC-4022 in 2.5 L fermentor,the enzyme activity increased from 14.67 U/m L to 144.89 U/m L,which was 9.88 times higher.The fermentation process of P.cyclopium CICC-4022 was of the growth-coupled type.The PC-Edex enzyme solution was purified by secondary tangential membrane filtration with a purification multiple of 3.68,a recovery of 62.52%and a specific activity of 11479.66 U/mg.The optimum temperature of PC-Edex was 60℃and the optimum p H was 5.0.PC-Edex was stable at 45-50℃and p H 3.0-7.0.PC-Edex specifically degradedα-1,6 glycosidic bonds and had no catalytic effect onβ-1,2 andβ-1,4 glycosidic bonds.The most suitable substrate was dextran-T70 and the most affinity substrate was dextran-T2000.K⁺,Na⁺,NH₄⁺,Tris and Urea all contributed to the promotion of PC-Edex activity,while the inhibitory effect of Ni²⁺,Zn²⁺,Co²⁺,Fe²⁺and EDTA increased with increasing concentration,with Cu²⁺being the strongest inhibitor.The whole genome of P.cyclopium CICC-4022 was sequenced by the third generation sequencing technology for the first time,and the function of its protein coding gene was annotated.The results showed that P.cyclopium CICC-4022 had strong life activities and rich carbohydrate transport and metabolism functions,and most of the proteases were mainly glyoxylase.In this study,a dextranase of GH49 family（PC-scaffold4.t4）was successfully annotated,which can act on glycosidic bonds and hydrolyze O-glycosyl compounds.The spatial structure of PC-Edex was obtained by homology modeling,and the binding site and binding free energy of PC-Edex with dextran were obtained by molecular docking and molecular dynamics,in which van der Waals force and electrostatic interaction energy were the main driving forces,and the polar solvent free energy was not conducive to the binding of dextran with PC-Edex.PHE402 has hydrophobic interaction,and ASP447 is a nucleophile,both of which are active sites in key sites.The optimized experimental conditions allow the regulation of the heavy average molecular weight（Mw）and molecular weight distribution（Mw/Mn）of high molecular weight dextran.Based on the Malhotra model,the regulation of PC-Edex degradation of high molecular weight dextran to a series of low molecular weight dextran can be achieved,and dextran with molecular weight less than 10 k Da can be obtained,and the molecular weight distribution is uniform,and the mass fraction of fragments with molecular weight less than 10 k Da can reach more than 94.56%,and the degradation rate can reach more than 98.96%.The appearance of dextran did not change during the enzymatic degradation process,and the Mark-Houwink and Einstein equations indicated that the chain conformation of dextran in aqueous solution was spherical.The molecular structure of dextran did not change after enzymatic degradation.TG showed no change in the thermal stability of dextran after enzymatic degradation,and NMR(¹H and ¹³C)and FI-TR indicated that theα-1,6 glycosidic bond remained as the main chain of dextran after enzymatic degradation.This study provides a technical tool for the enzymatic production of low molecular weight dextran and investigates the mode of interaction between PC-Edex and dextran at the molecular level,providing data to support subsequent industrial production and research.