【作者】 陈晟；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 角质酶是一种可以降解角质并产生大量脂肪酸单体的水解酶。角质酶是一种多功能酶,可水解可溶性酯、不溶性甘油三酯和各种聚酯,同时还能催化酸与醇的酯化、脂肪酸盐与醇的转酯化反应,因此在食品工业、化工工业等诸多领域都具有广泛的应用。近年来研究发现,角质酶可实现棉纤维的生物精练和合成纤维的生物改性,是推动纺织工业清洁生产的关键酶制剂。国外对真菌角质酶进行了深入的研究,但由于存在基因工程异源表达时表达量低和稳定性差等问题,至今没有实现工业化生产。与真菌角质酶相比,细菌角质酶具有催化效率高、基因工程异源表达技术成熟等方面的优势,但是国内外关于细菌角质酶的报道很少,迄今为止没有细菌角质酶基因鉴定的报道,因此细菌角质酶的研究受到限制。本论文完成了嗜热放线菌Thermobifida fusca角质酶的基因鉴定,实现了其在大肠杆菌中的高效表达及分子改造,主要结果如下:1.依次经过硫酸铵沉淀,Phenyl HP Sepharose FF疏水色谱,DEAE Sepharose FF阴离子交换色谱,从角质诱导的T. fusca发酵液中纯化得到电泳纯pNPB水解酶,比酶活由52.3 U/mg提高至398.6 U/mg,纯化倍数为7.6,回收率为9.3%。此外,通过硫酸铵沉淀,Phenyl HP Sepharose FF疏水色谱,CM Sepharose FF阳离子交换色谱,从重组Bacillus subtilis发酵液中纯化得到电泳纯Fusarium solani pisi角质酶。以F. solani pisi角质酶为对照品,苹果角质为底物,采用GC/MS分析,鉴定纯化得到的pNPB水解酶与F. solani pisi角质酶类似,能水解苹果角质并产生特征性产物,具有角质酶功能。2.纯化得到的T. fusca pNPB水解酶经肽指纹图谱鉴定,与数据库中Tfu₀882和Tfu₀883两个甘油三酯酶相符。通过N端测序得到纯化的蛋白N端前九个氨基酸为:ANPYERGPN。以T. fusca总DNA为模板,设计引物PCR得到编码Tfu₀882和Tfu₀883成熟蛋白的基因,并和具有PelB信号肽和6-His纯化标记的pET20b（+）质粒相连,使Tfu₀882和Tfu₀883在E. coli BL21（DE3）中过量表达。通过IPTG诱导,重组菌发酵上清液pNPB水解酶酶活Tfu₀882为52.5 U/mL,Tfu₀883为90.6 U/mL,分别是T. fusca诱导发酵液的4.37和7.56倍。采用Ni亲和柱对重组Tfu₀882和Tfu₀883进行分离纯化,纯化后重组蛋白达到电泳纯,重组Tfu₀882和Tfu₀883的pNPB水解酶比活力分别为223.1 U/mg、458.2 U/mg,并且两者均能水解角质并产生特异性产物,由此可确定,Tfu₀882和Tfu₀883是编码T. fusca角质酶基因。3.考察IPTG浓度和温度对重组大肠杆菌发酵生产Tfu₀883的影响,发现不添加IPTG和25°C恒温培养效果较好,发酵80 h培养基中pNPB水解酶酶活达130.5 U/ml。在该条件下发酵生产Tfu₀883,周质空间有大量重组蛋白积累,跨外膜转运成为重组蛋白向培养基中分泌的限速步骤。通过添加膜透性增强剂和改变环境条件加速重组Tfu₀883从周质空间向培养基中分泌。研究表明,在对数生长中期,添加100 mM甘氨酸、1 mM表面活性剂TDOC均可将酶活最高点从80 h提前至54 h;采取重组大肠杆菌在25°C发酵至24 h时将培养温度提高至37°C的变温策略对重组蛋白的分泌有促进作用,酶活最高点出现于62 h,比不变温的对照提前了18 h。4.重组Tfu₀882、Tfu₀883和F. solani pisi角质酶在水溶液中均以单体形式存在。重组Tfu₀882、Tfu₀883的最适温度为60°C,并具有良好的热稳定性;重组F. solani pisi角质酶的最适温度为40°C,热稳定性较差。三种角质酶的最适pH均为8.0,pH稳定范围为6.0-9.0。三种角质酶均能够水解可溶性酯、不溶性甘油三酯和聚酯,并倾向于水解短链底物,没有位置特异性。F. solani pisi角质酶对pNPB底物的亲和力最大,催化效率最高;F. solani pisi角质酶水解PET能力最强,Tfu₀883次之,Tfu₀882对PET仅有微弱水解。三种角质酶都没有界面活化现象,不需要二价金属离子作为辅助因子,Mn2+对三者有激活作用,Cr2+和Hg2+对三种角质酶有强烈抑制作用。Triton X-100和Tween 20抑制Tfu₀882和Tfu₀883的活性,对F. solani pisi角质酶无明显作用;SDS对三种角质酶均有竞争性抑制,Tfu₀882抑制最弱;TDOC对F. solani pisi角质酶有明显激活作用。重组Tfu₀882和Tfu₀883在甲醇、乙醇等多种有机溶剂中具有良好的稳定性,F. solani pisi角质酶在有机溶剂中的稳定性较差。5.以Streptomyces exfoliates脂肪酶晶体结构为模板,通过SWISS-MODEL软件模拟得到Tfu₀883和Tfu₀882结构模型。模型显示,T. fusca角质酶具有典型的α/β水解酶结构,Tfu₀883的活性中心由S170-H248-D216催化三角组成,Tfu₀882的活性中心由S188- H266-D234催化三角组成。T. fusca角质酶活性中心上方没有盖子结构,活性中心暴露于溶剂中。角质酶基因Tfu₀882和Tfu₀883在基因组中处在相邻位置,并处于两个不同的操纵子中,受到不同的调控。Tfu₀882和Tfu₀883在水解角质过程中没有协同作用。在结构模拟的基础上进行理性分析,并成功构建了角质酶突变体Tfu₀883-I218A、Tfu₀883-W195A/F249A和Tfu₀883-Q132A/T101A,催化性质研究表明,突变体对小分子底物的催化没有明显影响;突变体Tfu₀883-I218A和Tfu₀883-Q132A/T101A水解角质能力明显提高;突变体Tfu₀883-Q132A/T101A水解PET聚酯的能力也得到明显提高。更多还原

【Abstract】 Cutinase is a kind of hydrolase capable catalyzing the cleavage of ester bonds of cutin to release fatty acid. They display hydrolytic activity not only toward cutin but also a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Except for hydrolytic activity, cutinase also shows synthetic activity and transester activity. Therefore cutinase has been evaluated as a versatile lipolytic enzyme used in food and chemical industry. Rcently, it was found that cutinase has potential use in cotton bioscouring and synthetic fibers modification. Cutinase is the important enzyme in textile industry clean production.Fungal cutinase has been extensively investigated for many years. However, because of its low expression level and poor stability, recombinant fungal cutinase has not been produced in industry. In contrast to fungal cutinase, bacterial cutinase is more efficient in catalysis and easy to homologous expression. But there are just a few reports of bacterial cutinases. Most critically, no cutinase open reading frame has been identified in bacteria, thus it is impossible to study it in depth.In present paper, the gene of bacterial cutinase from Thermobifida fusca has been identified and high-efficient expressed in E. coli. And the recombinant T. fusca cutinase has also been molecular modified. The main results as follows:1. The pNPB hydrolase was purified from cutin-induced culture supernatant of T. fusca through ammonium sulfate precipitation, hydrophobic interaction （Phenyl Sepharose） and anion exchange （DEAE Sepharose） chromatography. The purified enzyme exhibited a high specific activity of 398.6 units/mg for pNPB. The purification fold is 7.6 and recovery yield is 9.3%. F. solani pisi cutinase was purified from supernatant of recombinant Bacillus subtilis through ammonium sulfate precipitation, hydrophobic interaction （Phenyl Sepharose） and cation exchange （CM Sepharose） chromatography. Two purified enzymes were used to hydrolyze apple cutin and the production were determined by GC/MS. It was found that both enzymes exhibited activity for cutin hydrolysis, yielding comparable fatty acid monomers.2. The purified T. fusca pNPB hydrolase was identified by Peptide Mass Fingerprinting Analysis, resulting in two significant matches, Tfu₀883 and Tfu₀882. The N-terminal amino acids were ANPYERGPN by sequencing. The gene encoding the mature form of Tfu₀882 and Tfu₀883 were amplified from T. fusca genomic DNA by PCR. The vector pET-20b（+） were chosen for expression, which contains a C-terminal His6-tag and a signal peptide PelB allowing the heterologously expressed proteins to be secreted. The constructed plasmid pET-20b/cutinase were expressed in E. coli BL21（DE3）. The pNPB hydrolase activity of recombinant Tfu₀882 and Tfu₀883 in culture supernatant were 52.5 U/ml and 90.6 U/ml, which were 4.37 and 7.56-fold higher than that of cutin-induced T. fusca cells. The recombinant enzymes were purified to homogeneity in a single step by nickel affinity chromatography and exhibited a specific activity of 458.21 units/mg for Tfu₀883 and 223.13 units/mg for Tfu₀882. Both recombinant enzymes can hydrolyze cutin to release monomers. Tfu₀883 and Tfu₀882 is the encoding gene of T. fusca cutinase3. The effect of IPTG induction concentration and temperature on the production of recombinant Tfu₀883 were determined. It was found that without IPTG induction and culture cell at 25°C for 80 h, the pNPB hydrolase activity in the culture supernatant was 130.52 U/ml. In addition, there was a lot amount of recombinant protein accumulated in periplasm. So that the transformation of recombinant protein out of outer membrane became the limiting-step of secretion. Adding additives and changing the environment condition to accelerate the secretion of recombinant protein is effective. The study showed that, adding 100 mM glycine and 1mM TDOC at mid-log growth phase could advance the peak of activity from 80 h to 54 h. Culturing the cell at 25°C until late-log growth phase, then increased the temperature to 37°C, in this way, the peak of activity can be advanced from 80 h to 62 h.4. Recombinant Tfu₀882, Tfu₀883 and F. solani pisi cutinase were all determined to be monomeric in solution. Tfu₀882 and Tfu₀883 exhibited an optimal temperature at 60°C and superior thermostability, while F. solani pisi cutinase displayed an optimal temperature at 40°C and significantly less stable. Furthermore, all three enzymes exhibited a pH optimum of about 8.0 and pH stability range is 6.0-9.0. Three enzymes can hydrolyze soluble synthetic esters, insoluble triglycerides and polyesters. They all prefered short chain substrate and showed no substrate position specificity. F. solani pisi cutinase exhibiting the highest affinity and the highest catalytic efficiency for pNPB. In addition, F. solani pisi cutinase exhibited the highest activity toward PET, while Tfu₀883 exhibited less activity. Three enzymes did not possess interfacial activation phenomenon, did not require divalent cations for activity. Mn active the three enzymes while Cr and Hg inhibited enzyme activity intensely. Triton X-100 and Tween 20 inhibited the activity of Tfu₀882 and Tfu₀883 while SDS inhibited all the three enzymes in competitive way. TDOC active the F. solani pisi cutinase greatly. Tfu₀882 and Tfu₀883 demonstrated great stability in many organic solvents such as mathol and ethnol while F. solani pisi cutinase is less stable in organic solvents.5. A homology model of Tfu₀882 and Tfu₀883 based on Streptomyces exfoliates lipase structure was constructed by the SWISS-MODEL homology-modeling web server. The predicted structure exhibits anα/βhydrolase fold of both two enzymes. In addition, it revealed a S170-H248-D216 catalytic triad for Tfu₀883 and S188- H266-D234 catalytic triad for Tfu₀882. Both two enzymes do not contain a lid insertion which resulting in a nucleophilic serine exposed to the solvent. The Tfu₀882 and Tfu₀883 genes are sequential in the genome and belong to separate operons. There is no synergism between the two enzymes in the degradation of cutin. Based on the structure model and rational analysis, the mutation enzymes Tfu₀883-I218A, Tfu₀883-W195A/F249A and Tfu₀883-Q132A/T101A were constructed. The study of catalysis properties showed that mutants exhibited no significant effect on low molecular weigh substrate. Mutants Tfu₀883-I218A and Tfu₀883-Q132A/T101A showed higher ability in hydrolyzing cutin. Tfu₀883-Q132A/ T101A exhibited obviously higher hydrolysis ability on PET polymer.更多还原