【作者】 成莉凤；

【导师】 刘正初；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2013， 博士

【摘要】 果胶酶是降解果胶类物质所需多种酶的总称。果胶酶的用途十分广泛，例如，用于饲料行业消除抗营养因子的添加剂，用于食品行业中果汁的提取和澄清处理，用于纺织业中麻类生物脱胶和棉纤维生物精炼以及用于造纸行业中的生物制浆。作为各行各业清洁生产的工具酶，碱性果胶裂解酶近年来受到越来越多的关注。本研究以基因组测序注释拥有14个果胶酶基因的麻类脱胶高效菌株DCE-01为基础，采用分子生物学、生物化学、酶学、结构生物学、生物信息学等方法，从基因水平和蛋白质酶学两个层面，开展果胶酶基因克隆及其表达产物的催化功能、酶学性质、分子结构等研究，获得如下结果：1.根据全基因组测序注释结果设计引物，从DCE-01菌株中成功克隆到14个果胶酶基因，包括11个果胶解聚酶基因、2个果胶酯酶基因和1个果胶脱乙酰基酶基因。其核苷酸序列与其他微生物来源的果胶酶基因核苷酸序列的一致性在71%-100%之间，其中，一致性高于95%的基因序列占1.8%，低于85%的基因序列占58.5%。DCE-01菌株所有14个果胶酶基因核酸序列之间的一致性在36.3%-84.2%之间；同样属于果胶解聚酶基因的核酸序列一致性同样在36.1%-84.2%之间，两个果胶酯酶的核酸序列一致性仅为42.8%。2.以果胶酶基因核苷酸序列为基础，生物信息学分析结果表明：DCE-01菌株14个果胶酶氨基酸序列与其他微生物的果胶酶氨基酸序列一致性在21%-100%之间，其中，氨基酸序列一致性在95%以上者占1.8%，一致性低于50%者占54.6%，与Dickeya dadantii3937的PelB（Pectate lyaseB）氨基酸序列比较，DCE-01菌株果胶解聚酶419在8个氨基酸位点上存在差异；DCE-01菌株的14个果胶酶氨基酸序列的一致性为16.4%-84.8%；MEGA聚类分析证明，该菌株果胶酶进化关系十分复杂。在14种果胶酶中，具有明显信号肽的果胶酶9种，没有明显信号肽的5种；定位在胞外的果胶酶10种，定位在胞内的2种，定位在细胞膜外膜的1种，无明确定位的1种；果胶酶分子量为30-90kDa；等电点为5.0-9.5。构建14个果胶酶基因的原核表达体系，平板水解圈法、酶活力测定法、SDS-PAGE电泳鉴定，有12个果胶酶基因获得成功表达，其中10个基因表达出具有生物活性的胞外果胶酶。经IPTG诱导，DNS法测定，有9个基因工程菌株的胞外果胶解聚酶活力为0.08-164.9IU/mL。深入研究结果表明，果胶解聚酶RP65、5P8、SH8和4J4的最适反应温度分别为50°C、45°C、50°C和55°C；最适pH均为8.5；热稳定温度分别为40°C、40°C、35°C和45°C；最适Ca²⁺终浓度分别为0.5mmol/L、0mmol/L、2.0mmol/L和1.0mmol/L；最适底物分别为酯化度20%-34%的橘子果胶、聚半乳糖醛酸钠、酯化度20%-34%的橘子果胶和酯化度≥85%的橘子果胶。3. Pel419的最适反应温度为50°C；保温1.0h，酶活稳定温度≤45°C；最适pH为9.0；稳定pH为8.5-10.0；Ca²⁺、Zn²⁺和NH₄⁺促进酶活力，Ca²⁺的最适作用终浓度为2.0mmol/L，Fe³⁺、Pb²⁺和EDTA严重抑制酶活力；聚半乳糖醛酸钠为最适底物。PelG403最适反应温度为55°C；保温1.0h，酶活稳定温度≤60°C；最适反应pH为9.5；稳定pH为9.0-10.0；Ca²⁺能最大幅度提高酶活力，其最适作用终浓度为1.5mmol/L，Pb²⁺和EDTA能严重抑制酶活力；苹果果胶为最适底物。诱导基因工程菌株pEASY-E1-243/BL21表达有生物活性的胞外果胶酯酶，以高度酯化橘子果胶（DE≥85%）为底物，测定其发酵液酶活为1.5IU/mL，是原始菌株DCE-01的22.4倍。综上所述，本研究成功从DCE-01菌株中克隆出全基因组测序注释的14个果胶酶基因。通过系统研究果胶酶基因核苷酸序列及果胶酶分子结构、酶学性质、催化功能，证实DCE-01菌株果胶酶基因资源具有极为丰富的多样性。初步鉴定出在生物质加工过程中具有重要工业化应用前景的工具酶3种，为阐明麻类生物脱胶的作用机理提供科学依据，为发掘优异生物质加工用果胶酶资源奠定基础。更多还原

【Abstract】 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.更多还原