Experssion,Characterization And Application Of Marine Chitosanases

Chitooligosaccharide（COS）has better water solubility compared with chitosan.Because of its lower degree of polymerization,it can be easily absorbed and utilized.At present,COS are mainly prepared by chemical,physical and enzymatic methods.Enzymatic degradation of chitosan to produce COS is not only in line with the green production advocated by the current national appeal,but also can specifically degrade chitosan into COS with desired DP.Therefore,it is one of the main methods to prepare COS with controllable degree of polymerization.However,currently only a limited number of chitosanases have been characterized.Few studies towards marine chitosanases have been carried out.It is hoped that current study can provide relevant information for marine chitosanase,and provide a new direction for seeking to produce chitosanase with specific characteristics,thereby reducing the price of current commercial chitosanases.The main content of this paper is as follows:1.Expression,properties and application of marine chitosanase SN1-CSN from Streptomyces niveusIn this paper,the marine chitosanase SN1-CSN from Streptomyces niveus was expressed in E coli and purified by nickel column,By SDS-PAGE analysis,the predicted molecular weight of Sn1-CSN based on amino acid sequence was 29.8 k Da.DNS method was used to determine the optimal p H,p H stability,optimal temperature,thermal stability and the effects of metal ions on enzyme activity.The optimum p H of Sn1-CSN is 6.0,and the stability of Sn1-CSN is relatively high in the range of p H 4-11.The optimum temperature is 50°C;Sn1-CSN can maintain excellent thermal stability at 45°C.The enzyme activity of Sn1-CSN was significantly increased after the addition of Cu²⁺,and the activity of Sn1-CSN was inhibited by Fe³⁺.The apparent k_mand k_catvalues of Sn1-CSN were 1.8 mg/m L and 88.3 s^-1,respectively.Through TLC,HPLC and MS analysis,it can be seen that the main hydrolysis product of chitosan（degree of deacetylation,DDA>94%）catalyzed by Sn1-CSN is chitobiose,without monosaccharide production,demonstrating that Sn1-CSN is an endo chitosanase.Sn1-CSN into partially acetylated COS with DP 2-15,and the composition was relatively complex.According to the phylogenetic tree,Sn1-CSN shares the same branch with SACTE＿5457from Streptomyces sp.Sirex AA-E.The two conserved glutamic acid（Glu 40）and aspartic acid（Asp 58）residues are regarded as catalytic residues in Sn1-CSN.From the results of molecular docking,it can be seen that the interaction between Sn1-CSN and chitobiose is weaker than that of chitotriose.2.Expression,properties and application of marine chitosanase SPAMC-CSN from Streptomyces sp.PAMC 26508In this paper,the marine chitosanase SPAMC-CSN from Streptomyces sp.PAMC 26508was expressed in E coli and purified by nickel column,By SDS-PAGE analysis,the predicted molecular weight of Sn1-CSN based on amino acid sequence was 29.37 k Da.The optimum p H of SPAMC-CSN is 7.0,which is a neutral enzyme;it maintains relatively high stability in the range of p H 4-11;the optimum temperature is 45°C;SPAMC-CSN can maintain good thermal stability at 50°C;the enzyme activity of SPAMC-CSN is significantly improved after Cu²⁺is added.Fe³⁺and Zn²⁺can inhibit the activity of SPAMC-CSN;the apparent k_mand k_catvalues of SPAM-CSN are 0.724 mg/m L and 88.3 s^-1,respectively.TLC,HPLC and MS analysis showed that the main hydrolysis products of chitosan catalyzed by SPAMC-CSN（degree of deacetylation,DDA>94%）were chitobiose,chitotriose and chitotetraose.SPAMC-CSN into partially acetylated COS with DP 2-10.According to the phylogenetic tree,SPAMC-CSN has high sequence identity with Streptomyces sp.SACTE＿5457 and Sc Csn A.The catalytic residues of SPAMC-CSN are glutamic acid（Glu 40）and aspartic acid（Asp 58）residues.From the results of molecular docking,it can be seen that the interaction between SPAMC-CSN and chitobiose is weaker than that of chitotriose.