Cloning And Expression Of Pectinase Genes From DCE-01Strain And Their Diversity

Pectinase is a heterogeneous group of related enzymes that hydrolyze the pectin. Pectinase is usedwidely in various industries, such as in feed industry as additives for anti-nutrients elimination, in foodindustry for fruit juice extraction and clarification, in textile industry for process of cotton fiber and bastfibers biodegumming, and in the papermaking industry for bio-pulping. As one of the most importantcatalytic agents in all walks of life, alkaline pectate lyases have been paid more and more attention inrecent years.From previous studies, fourteen pectinase genes had been annotated in the genomic sequence ofDCE-01strain, an efficient strain for bast fibers biodegumming. Based on this preliminary study, withthe methods of molecular biology, biochemistry, enzymology, structural biology, bioinformatics and soon, pectinase genes cloning and catalytic function, enzymatic properties and molecular structure of theirexpression products were studied. Main results were as follows:1. Primers were designed by the result of whole genome sequencing annotation, and fourteenpectinase genes were obtained. Fourteen pectinase genes were divided into three groups which wereeleven pectin depolymerase genes, two pectin methyl esterase genes, and one pectin acetyl esterase gene.Nucleotide sequence identities of pectinase genes from DCE-01strain and other microbiology were71%-100%. Ratio of pectinase genes with identity higher than95%was1.8%, and that with identityless than85%was58.5%. Nucleotide sequence identities among the fourteen pectinase genes fromDCE-01strain were36.3%-84.2%. Identities of the eleven pectin depolymerase genes were36.1%-84.2%, and the identity of the two pectin methyl esterase genes was only42.8%.2. Based on the pectinase genes nucleotide sequences, the bioinformatics analysis results showedthat amino acid sequence identities of the pectinase from DCE-01strain and other microbiology were21%-100%. Ratio of pectinase with identity higher than95%was1.8%, and that with identity less than50%was54.6%. Comparing the pectin depolymerase419from DCE-01strain with the PelB from D.dadantii3937, they were obviously different in eight amino acid sites. MEGA cluster analysis provedthat evolution relationship of the fourteen pectinase from DCE-01strain was very complex. Amongfourteen pectinase, nine pectinase had obvious signal peptides, and five pectinase had no apparent signalpeptides. Ten pectinase located in extracellular, two pectinase located in intracellular, one pectinaselocated in outer membrane of the cell membrane, and one pectinase had no clear position. Themolecular weights of pectinase were30-90kDa, and the isoelectric points were5.0-9.5. Prokaryoticexpression systems were constructed to express the fourteen pectinase genes. By plate hydrolysis circles,pectinase activity assay and SDS-PAGE analysis, the results showed that twelve pectinase succeed inexpressing, and ten extracellular pectinase had catalytic activity. Detected by DNS method, theextracellular pectin depolymerase activity secreted by nine genetic engineering strains induced by IPTG,were0.08-164.9IU/mL. The optimal reaction temperatures of depolymerase RP65,5P8, SH8and4J4were50°C,45°C,50°C and55°C respectively. Their optimal reaction pHs were8.5. Theirthermostable temperatures were40°C,40°C,35°C and45°C in sequence. Their optimal Ca²⁺ concentrations were0.5mmol/L,0mmol/L,2.0mmol/L and1.0mmol/L. Their optimal substrates werecitrus pectin esterified20%-34%, polygalacturonic acid sodium salt, citrus pectin esterified20%-34%and citrus pectin esterified≥85%, respectively.3. The optimal reaction temperature of the Pel419was at50°C and was stable at no more than45°C after being incubated for60min. The optimal reaction pH of the Pel419was pH9.0and wasstable at pH8.5-10.0. The Pel419activity was activated by Ca²⁺, Zn²⁺and NH₄⁺, and the maximalactivity of Pel419was obtained at Ca²⁺concentration of2.0mmol/L. The optimum substrate for Pel419was polygalacturonic acid sodium salt. The optimal reaction temperature of the PelG403was at55°Cand was stable at no more than60°C after being incubated for60min. The optimal reaction pH of thePelG403was pH9.5and was stable at pH9.0-10.0. The PelG403activity was activated by Ca²⁺andinhibited seriously by Mn²⁺, Pb²⁺and EDTA. The maximal activity of PelG403was obtained at Ca²⁺concentration of1.5mmol/L. The optimum substrate for PelG403was pectin from apple.pEASY-E1-243/BL21was induced by IPTG to express extracellular pectin methyl esterase. With highmethoxyl citrus pectin （DE≥85%） as substrate, the pectin methyl esterase activity secreted by thegenetic engineering strain was1.5IU/mL,22.4times higher than that from the original DCE-01strain.The above results fully reveal fourteen pectinase genes were cloned from DCE-01strain, and thatpectinase expressed by the pectinase genes from DCE-01strain had very rich diversity in molecularstructure, enzyme properties, and catalytic function. Moreover, three important tool enzymes potentialfor industrial application in the process of biomass were identified primarily. This study may providescientific basis to elucidate the mechanism of bast fibers biodegumming, and lay foundation to explorethe excellent pectinase resources for biomass process.