Heterologous Expression And Enzymatic Properties Of A Novel Chitin Deacetylase

Chitin deacetylase(CDA)is an enzyme that removes acetyl groups of chitin to produce chitosan and its derivatives.The product chitosan and its derivatives can be applied in the fields of biomedicine,food processing,etc.,and have broad application prospects.Compared to the hot alkali chemical method,the use of CDA to prepare chitosan has the advantages of low costs,and uniform product quality.In this thesis,the high-yielding CDA of Rhodococcus equi(ReCDA)screened from soil is chosen to be the research objective,the preliminary analysis of the enzymatic properties and substrate preference of ReCDA is performed by heterologous expression,optimization of enzyme production conditions and protein purification.ReCDA was heterologously expressed in E.coli BL21(DE3),E.coli Rosetta,Saccharomyces cerevisiae BY4 741 and Pichia pastoris GS115 without CDA activity using genetic engineering technology.Meanwhile,the enzyme-producing conditions of each heterologous expression strain were optimized.The optimal conditions for the expression of ReCDA in E.coli BL21/pET28a(+)-ReCDA-His(6)and E.coli Rosetta/pET28a(+)-ReCDAHis(6)are optical density value(OD600)in the range of 0.6 to 0.7,and 0.15 mM IPTG induction for more than 48 h at 22℃.The enzyme activity reached 119.58 U/mL,but its protein expression level was low by SDS-PAGE analysis.The optimal conditions for the expression of ReCDA in Saccharomyces cerevisiae BY4741/pYES2.0-ReCDA-His(6)is the OD660 of 1.0,and 0.2%methanol induction for 90 h at 30℃.The enzyme activity reached 17.25 U/mL,and ReCDA protein bands are obvious by SDS-PAGE analysis.The optimal conditions for the expression of ReCDA in Pichia pastoris GS115/pPIC3.5K-ReCDA-His(6)and Pichia pastoris GS115/pPIC9K-ReCDA-GST are the OD660 in the range of 1.0 to 2.0,and 2%methanol induction for 90 h at 30 ℃.The enzyme activity reached in the range of 75.85 U/mL to 85.68 U/mL.Compared with ReCDA heterologously expressed in E.coli and Saccharomyces cerevisiae,the highest protein expression was found in Pichia pastoris heterologous expression strain.The effects of Ni-NTA affinity chromatography,anion exchange chromatography and GST-glutathione affinity chromatography on the purification of ReCDA were investigated.The purification conditions of ReCDA are optimized,and finally the ReCDA protein is purified,and its relative molecular mass is about 33.8 kDa.Enzymatic analysis of the purified ReCDA showed that the optimal pH was 7.5 and the optimal temperature was 35℃.In addition,1 mM Zn2+showed obvious inhibitory effect on enzyme activity.Furthermore,the substrate preference of ReCDA was further analyzed.ReCDA showed significant deacetylation activity in colloidal chitin,ethylene glycol chitin,chitin oligosaccharide,chitosan(DAs,85%)and some acetyl-containing amino acids.This is the first time to analyze the enzymatic properties of ReCDA through heterologous expression and protein purification.It not only enriches the research on the chitin deacetylase family,but also provides a reference for the industrial production and application of bioenzymatic production of chitosan and its derivatives.