【作者】 郝之奎；

【导师】 廖祥儒；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 几丁质是自然界含量仅次于纤维素的碳水化合物和仅次于蛋白质的含氮有机物,其水解产物N-乙酰-D-氨基葡萄糖、几丁寡糖及其衍生物在食品、医药、农业、化工等行业应用极为广泛。几丁质酶能催化水解N-乙酰葡萄糖胺基团糖苷键,降解几丁质,生成几丁寡糖、几丁二糖或N-乙酰-D-氨基葡萄糖等,因此,研究几丁质酶基因资源、产酶发酵及基因表达调控等具有一定的理论意义和潜在的应用价值。论文从废弃池塘底泥中筛选获得一株几丁质酶高产细菌,对该菌进行了鉴定,深入研究了其产酶条件、酶的分离纯化和酶学性质、几丁质酶的产生机理、几丁质酶的克隆与表达以及几丁质酶在水解几丁质方面的应用等,主要结果如下:1)利用透明圈法,从采自4省市的180余份土壤、枯枝、昆虫、甲壳动物尸体等样品中筛选出56株合成几丁质酶的细菌,其中有5株产酶能力较强;选择其中一株几丁质酶产量较高的菌株,从形态学、生理生化、脂肪酸组成、醌类、G+C含量及16S rRNA等方面进行了鉴定。确定该菌属变形菌纲（Proteobacteria）、奈瑟菌目（Neisseriales）、奈瑟菌科（Neisseriaceae）下的一个新属新种,命名为Chitinolyticbacter meiyuanensis SYBC-H1,并提交至中国普通微生物菌种保藏管理中心（菌株编号CGMCC 3438）和美国典型微生物菌种保藏中心（菌株编号ATCC BAA-2140）保藏。2)利用单因素实验和响应面分析法对C. meiyuanensis SYBC-H1的发酵培养基及培养条件进行了优化。利用单因素实验筛选出对几丁质酶产量影响较大的10个因子,利用Plackett-Burman Design实验筛选出影响几丁质酶合成的关键因子:菊芋粉、尿素及硫酸钠,再利用Central Composite Design建立模型,获得了最佳发酵培养基组成（g/L）:菊粉3.55,尿素3.10,几丁质3.80,Na₂SO₄ 0.32,K₂HPO₄·3H₂O 0.70,KH₂PO₄ 0.30,MgSO₄·7H₂O 0.5,FeSO₄·7H₂O 0.01。利用Central Composite Design对几丁质酶合成的培养条件进行了优化,最优结果为:转速230 r/min,起始pH 6.4,温度26℃;在此条件下最大酶活为5.16 U/mL,几丁质酶活力比优化前提高6.21倍。3)通过硫酸铵分级盐析、DEAE-cellulose阴离子交换层析及Sephadex G-100分子筛层析三个步骤,得到一种分子量约为42 kDa电泳纯的几丁质酶,并在此基础上研究了几丁质酶的酶学性质。结果显示,C. meiyuanensis SYBC-H1几丁质酶的最适温度为40℃;最适pH为6.5;几丁质酶在45℃下比较稳定,55℃以上稳定性迅速下降;巯基乙醇（10 mmol/L）及EDTA（10 mmol/L）能有效提高几丁质酶的稳定性;Zn²⁺、Cu²⁺、Mn²⁺、Fe²⁺、Fe³⁺（10 mmol/L）对丁质酶有不同程度的抑制作用,Na⁺、K⁺对几丁质酶有激活作用。该几丁质酶的Km为22.96 mmol/L,Vmax为9.066 U/mg。4)研究了C. meiyuanensis SYBC-H1在优化培养基与基础培养基发酵过程中胞内外酶活变化与氧化胁迫和抗氧化酶的关系。结果表明,在菌体高产几丁质酶时,其胞内外丙二醛、过氧化氢含量明显较高,Vc含量、超氧化物歧化酶和过氧化氢酶活性等明显上升;说明C. meiyuanensis SYBC-H1几丁质酶基因表达可能受氧化还原调控。5)在考察碳源对酶活的影响时发现,以果糖为碳源的酶活远高于以葡萄糖为碳源时的酶活,克隆了C. meiyuanensis SYBC-H1 ABC转运系统中ATP酶、细胞膜周质糖结合蛋白及氨基糖苷转运蛋白基因,研究了其在C. meiyuanensis SYBC-H1发酵产几丁质酶中的作用。结果表明,C. meiyuanensis SYBC-H1的ATP酶基因全长1236 bp,编码377个氨基酸残基;细胞膜周质空间糖结合蛋白基因全长1329 bp,编码292个氨基酸残基;氨基糖苷转运蛋白基因全长1027 bp,编码340个氨基酸残基。这些基因的表达与其胞外几丁质酶活性增高相关,说明了ABC转运系统在该菌几丁质酶合成中的可能作用,为该菌进一步的分子改造构建生物工程菌提供了理论依据。6)根据与C. meiyuanensis SYBC-H1遗传距离较近的微生物已经克隆出的几丁质酶基因设计17对引物,从C. meiyuanensis SYBC-H1基因组中克隆得到C. meiyuanensis SYBC-H1几丁质酶基因CHI1和CHI2,基因全长分别是497 bp和1151 bp,分别编码166 Aa残基和383 Aa残基的几丁质酶。通过Swiss-MODEL分别研究了其三维结构,结果表明,CHI1主要由5个α螺旋和3个β折叠组成;而CHI2则主要由12个α螺旋组成,无β折叠结构。构建了CHI2基因的表达载体pET28（a）-CHI2并在E.coli BL21中进行了表达。7)通过高效液相色谱（HLPC）及质谱（MS）鉴定了几丁质的水解产物,并对C. meiyuanensis SYBC-H1产几丁质酶降解几丁质的条件进行了研究。结果表明,在几丁质酶催化下,几丁质水解的主要产物是N-乙酰氨基葡萄糖,几丁二糖的量极小;100目的几丁质粉粒有利于几丁质酶的水解,但30-70目的几丁质粉粒粒径的大小对几丁质的水解没有明显影响。同时还利用麦夸特法（Levenberg-Marquardt）及通用全局优化法建立了几丁质酶水解几丁质模型,结果显示:温度37.5℃,pH为7.0时水解前13.5 h水解效率最高,此时水解液中糖浓度为90.4μmol/L。24 h内最大水解率为79%。更多还原

【Abstract】 Chitin, an insoluble linearβ-1,4-linked polymer of N-acetylglucosamine （GlcNAc）, is frequently regarded as the second most abundant carbohydrate after cellulose in nature,and the second most abundant nitrogen organic after protein. N-acetylglucosamine（GlcNAc）, chitin oligosaccharide and its derivatives have a wide range of applications in industries of food, medical, agriculture, chemistry. As a group of enzymes capable of degrading N-acet- ylglucosamine monomers, chitinases hydrolyze chitin into chitin oligosaccharide, chitin disaccharide and N-acetylglucosamine.This dissertation reported a high chitinase-producing bacterium isolated from abandoned pond sediment. According to various characteristics, the bacterium was identified as a new species. The high chitinase-producing bacterium was deeply explored and researched, including strain isolation, optimization of chitinase-producing conditions, purification and characterization of chitinase, biosynthesis mechanism of chitinase, cloning and expresssion of chitinase gene, and its application in hydrolyzing chitin and shrimp by chitinase. The results were as follows:1)The 56 bacteria producing chitinase were isolated from 180 samples in four provinces through see-through circle way, including soil, insect, stricks and crustacean cadavers and so on. Among them, five bacteria have high chitinas-producing ability and one strain was selected and identified as a new species. According to the experiments of physiology and biochemistry, fatty acid composition, quinines, G+C content and 16S rRNA, it was named C. meiyuanensis SYBC-H1. The 16S rRNA has been submitted to GenBank （GQ981314）. This bacterium was preserved in the China General Microbiological Culture Collection Center （CGMCC 3438）and American Type Culture Collection（ATCC BAA-2140）.2)The submerged culture conditions and medium components of C. meiyuanensis SYBC-H1 were optimized. Effects of various carbon sources, nitrogen sources, metal ions and other factors were investigated firstly and ten important factors of chitinase production were screened. Furthermore some key factors for chitinase production were determined through Plackett-Burman design. The optimized medium （g/L） using central composite design were urea 3.1, inulin 3.55, chitin 3.80, Na₂SO₄ 0.32, K₂HPO₄·3H₂O 0.70, KH₂PO₄ 0.30, MgSO₄·7H₂O 0.5, FeSO₄·7H₂O 0.01. And then the optimum culture conditions were investigated through Central Composite Design,and the results optimum fermentration condition as rotate speed 233 r/min, initial pH 6.44 and temperature 26.21℃. The maximum enzyme activity was 5.16 U/mL while the tested enzyme activity was 5.20±0.32 U/mL. Meanwhile, the effects of cell age, inoculums size and surfactant on production of chitinase were studied.3)The crude chitinase was purified through three effective steps involving （NH₄）2SO₄ salt precipitation, DEAE-cellulose anion-exchange chromatography and Sephadex G-100 gel filtration. Pure electrophoresis grade chitinase was obtained. Enzymatic properties were studied,and its optimum temperature is 40℃, which aldso found that the enzyme activity decreases rapidly with rising temperature especially above 43℃. The chitinase activity with a wider range pH from 3.5 to 9.0 and the optimum pH of 6.5. Mercaptoethanol and EDTA effectively improved the stability of chitinase. Zn²⁺,Cu²⁺,Mn²⁺,Fe²⁺ and Fe（3+） have different inhibition degrees on chitinase , while Na⁺ and K⁺ stimulated it.4)The physiological characteristics of C. meiyuanensis SYBC-H1 during the chitinase production were investigated. It was found that secretion of chitinase and biomass had certain correlation. When the strain cultivated in the optimal medium, the oxidation level was relatively high, the contents of malondialdehyde, H2O2 and Vc were increased, and the activities of SOD and CAT were enhanced. These indicated that the strain was under oxidative stress during the chitinase production period.5)By observing the effect of carbon sources on enzyme activity, it was found that fructose as a kind of carbon source is far more effective than glucose. The cloning and expression experiment of chitinase gene enables us to clone and testify the ABC transportors system including ATPase, periplasmic sugar-binding protein and aminoglycoside transport protein. These offered theoretical base for further steps of molecule modification of this bacteria to design and construct bioengineering one.6)Two chitinase genes（CHI1,CHI2） were cloned and their full-length were 497 bp and 1151 bp. Three-dimensional structures were studied with Swiss-MODEL. E. coli expression vector pET28（a）-CHI2 was constructed and expressed into E. coli BL21 successfully. SDS-PAGE showed chitinase had significant expression but its activity was low, indicating that the expressed protein existed in the form of inclusion bodies.7)According to mass spectrometry （MS） and high performance liquid chromatography （HLPC） identification of hydrolysis products of chitin, there are mainly N acetyl glucosamine and few chitobiose. Chitinase degradation of chitin and dried small shrimps were investigated. Study showed that temperature, time, pH, beta mercaptoethanol, metal ions and oxygen supply affected the hydrolysis of chitin. Particle size of 100 hole of chitin powder was in favor of chitinase hydrolysis, but 30-70 chitin powder did not significantly affect. At the same time also we used Levenberg-Marquardt and general global optimization method to establish the chitinase hydrolysis chitin model, and the results showed that when the temperature was 37.5℃, pH 7.0 and the hydrolysis time 13.5 hours, the highest efficiency was reached, and sugar concentration was 90.4μmol / L.更多还原