Study On The Enzymatic Properties Of Pectin Lyase PelA And The Antibacterial Activity Of Pectin Oligosaccharides

Pectin oligosaccharide(POS)is the product of pectin hydrolysis,which has a variety of biological activities such as anti-obesity,anti-diabetes,antibacterial,anti-infection,and has important applications in medicine,food,cosmetics and other fields.Pectin oligosaccharides are mainly prepared by enzymatic degradation of pectin.Pectinase is a commonly used enzyme.Pectin lyase can degrade pectin without damaging ester groups,and is an important enzyme for producing pectin oligosaccharides.However,it is currently faced with the problems of few types and insufficient enzymatic properties for industrial applications.Therefore,exploring novel pectin lyase is still the current research focus.The main research contents and results of this paper are as follows:(ⅰ)Bioinformatics analysis:the laboratory obtained a marine bacterium,the scientific name of which is Echinicola pacifica,and found that it can degrade pectin.A gene of pectin lyase Pel A(GeneBank:NZ_KB89073 8)was found in its gene bank.After phylogenetic tree analysis of the pectin lyase gene,it was found that the enzyme belongs to the PL10 family.The gene sequence was compared with the gene sequence of other enzymes in the PL 10 family,The result shows that the sequence similarity is low and has some novelty.Subsequently,the amino acid sequence of the enzyme was analyzed by signal peptide and transmembrane structure prediction.The signal peptide was removed when the primer was designed.After the vector was constructed,it was induced to express in E.coli BL21(DE3).(ⅱ)Protein purification and enzymatic properties study:Pectin lyase PelA was purified by Ni-IDA affinity chromatography column,and purified by elution at 0.5 M imidazole concentration to obtain high purity protein with molecular weight of about 49 kDa.First of all,the specificity of its substrate was investigated.The results showed that PelA was a pectin lyase.This studied the enzymatic properties of PelA,and found that the optimum pH of the enzyme was 8.0,and the optimum temperature was 40℃.There were 11 metal ions that could inhibit the activity of PelA,and only Ca2+ could promote its activity,indicating that it was Ca2+ dependent.(ⅲ)Optimization of conditions and product analysis of PelA’s degradation of polygalacturonic acid:the product of PelA’s degradation of polygalacturonic acid(PGA)could not pass TLC analysis,so the conditions of PelA’s enzymatic hydrolysis of PGA were optimized by corresponding methods,and the yield was increased by 26%after optimization.The product was used for thin layer chromatography(TLC),ethanol precipitation,carbohydrate polyacrylamide gel electrophoresis(C-PAGE)Gel permeation chromatography(GPC)analysis showed that the hydrolysate was a high molecular oligosaccharide.It is verified that PelA can degrade citrus pectin,and many citrus pectins have antibacterial activity.Therefore,we extracted pectin from the commercial orange peel and degraded it,and then analyzed its product using the above method,and concluded that the product is a macromolecular pectin oligosaccharide.(ⅳ)Evaluation of the antibacterial activity of orange peel pectin oligosaccharide:In order to explore the biological activity of orange peel pectin oligosaccharide,the antibacterial activity of Staphylococcus aureus,Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa,Staphylococcus epidermidis,Bacillus subtilis and other bacteria were evaluated respectively,The bacteriostatic results showed that the inhibition effect of 6.25 mg/mL orange peel pectin oligosaccharide on gram-positive bacteria was obvious,and the inhibition effect on gram-negative bacteria was poor.The inhibition rate decreased with the decrease of the concentration of pectin oligosaccharide,which could provide theoretical support for industrial application.