几丁质及高附加值产物酶法生产关键技术研究博士论文

论文分类号： 2 学校代码： 10708 学 号： BS0808008 博博 士士 学学 位位 论论 文文 Dissertation for Doctors Degree 几丁质及高附加值产物酶法生产关键技术研究几丁质及高附加值产物酶法生产关键技术研究 STUDIES ON THE KEY TECHNOLOGIES TO PRODUCE CHITIN AND DERIVATIVES WITH HIGH VALUE BY ENZYMATIC METHODS 缑敬轩 指导教师姓名：董文宾 教授 学 科 名 称：应用化学 论文提交日期：2012 年 11 月 论文答辩日期： 学位授予单位： 答辩委员会主席： 评阅人： 申请 工学博士 学位论文 论文题目： 几丁质及高附加值产物酶法生产关键技术研究几丁质及高附加值产物酶法生产关键技术研究 学科学科门类门类：工学 一级学科：一级学科：化学工程与技术 培养单位：培养单位：化学与化工学院 博士生：博士生：缑敬轩 导导 师：师：董文宾 教授 2012 年 11 月 STUDIES ON THE KEY TECHNOLOGIES TO PRODUCE CHITIN AND DERIVATIVES WITH HIGH VALUE BY ENZYMATIC METHODS A Dissertation Submitted to Shaanxi University of Science and Technology in Partial Fulfillment of the Requirement for the Degree of Doctor of Engineering Science By Jingxuan Gou Dissertation Supervisor: Professor Wenbin Dong Nov., 2012 I 几丁质及高附加值产物酶法生产关键技术研究几丁质及高附加值产物酶法生产关键技术研究 摘摘 要要 几丁质是自然界产量仅次于纤维素的可再生性多聚物。自然界在不断 产生大量几丁质的同时，微生物也合成了大量能够降解几丁质的不同性质 的酶。几丁质在自然界的广泛分布，意味着能够分解利用几丁质及其类似 物的微生物也有着广泛的分布。环境的多样性、微生物种类的多样性为人 类筛选不同性质不同要求的几丁质及其衍生物降解酶提供了可能性。 近年来几丁质及其脱乙酰基产物壳聚糖和他们的寡糖，几丁寡糖和壳 寡糖，在诸如食品、制药、材料科学、微生物学、组织工程、纳米材料等 很多领域得到广泛的应用。因此对几丁质及其相关产物形成巨大的需求， 但是目前无论几丁质的工业化生产，还是几丁质脱乙酰基生产壳聚糖或生 产几丁寡糖、壳寡糖，均以化学方法为主，几乎都要用到强酸强碱，对环 境造成极大的污染。因此迫切需要开发新的绿色生产工艺。 采用生物学或酶学方法制备几丁质、壳聚糖及其寡糖具有生产条件温 和可控，对环境污染少的优点，因此是替代化学方法的理想技术，也是目 前的研究重点。目前几丁质的生产主要是从虾蟹壳中采用盐酸和氢氧化钠 分别脱除碳酸钙和蛋白质的办法，尽管有了一些改进，但是仍需要使用强 酸或强碱。另一方面，科研人员从自然界，特别是海洋环境中已经筛选出 很多和几丁质分解有关的酶，如几丁质酶（EC 3.2.1.14） 、几丁质脱乙酰基 酶(CDA; EC 3.5.1.41)和壳聚糖酶(EC 3.2.1.99)等， 但是都普遍都存在酶活力 不够高和酶产量低的问题， 因此多处于实验室阶段， 尚未应用于工业生产。 基于此，本研究首先尝试采用蛋白酶和有机酸分别脱除虾壳中蛋白质和碳 酸钙的办法生产几丁质，然后从与海洋环境差异巨大的秦岭山区不同生境 中采集土样，从中分离新型几丁质酶和几丁质脱乙酰酶高产菌株，并对其 培养基和发酵条件进行优化。 本研究尝试采用柠檬酸脱钙、胃蛋白酶去除蛋白质的工艺从虾壳中提 取几丁质。首先以灰分的含量为指标，通过单因素试验确定柠檬酸浓度和 处理时间的最佳参数，即浓度 12%的柠檬酸溶液处理 13 小时。该处理得到 的几丁质灰分含量为 1.5%，达到工业级的标准（3%） ，接近食品级标准 （1%） 。胃蛋白酶去除虾壳中蛋白质的研究中，采用凯氏定氮法测定蛋白 质的含量。在考察胃蛋白酶的用量（U/g） 、时间（h） 、温度（）和 pH 等 单因素基础上，利用响应面优化方法的 Box-Behnken 设计，以脱除蛋白质 II 的量为响应值， 构建了酶的用量、 时间和温度的三维响应曲面模型。 ANOVA 分析和实际验证证明该模型准确可靠。通过该模型推测出最佳处理参数为 胃蛋白酶使用量为 700 U/g、反应温度 36.09和反应时间 5h，蛋白质的脱 除率达到 8.47%，占总蛋白质含量的 27.89%， 。蛋白质去除率偏低，推测与 虾壳几丁质的微观结构有关。可以通过先脱钙再脱蛋白或者进行两次脱钙 和脱蛋白的方法提高蛋白质的去除效果。 优良的菌种是微生物发酵的基础和关键，一直是微生物降解几丁质研 究的重要内容之一。但目前筛选到的几丁质酶和几丁质脱乙酰酶生产菌株 均存在酶活力不够高和稳定性差的问题，因此仍处于实验室阶段，距离工 业化应用还有很大的差距。本研究从秦岭山脉采集土壤样品，经过富集培 养后，从中筛选几丁质酶和几丁质脱乙酰酶高产菌株。几丁质酶高产菌株 的初筛选是利用以胶体几丁质为唯一碳源的筛选培养基进行培养，从中挑 出 100 株产生较大透明圈的菌株，根据 D 值（透明圈与菌落直径的比值） 大小筛选出 20 株产酶能力较强的菌株。复筛选采用液体摇瓶发酵，DNS 法测定酶活力的方法筛选出产酶能力最高的 3 株细菌，编号分别为 Z4、F9 和 D5-23。其中菌株 Z4 的酶活最大，达到了 2.3 U/mL。菌体形态观察和 16S rDNA 序列分析表明菌株 Z4 属于微杆菌属(Microbacterium)。通过优化 培养基的组成和培养条件，其产酶能力得到极大的提高。首先以发酵液中 的酶活为指标，通过单因素实验确定其产几丁质酶的最佳碳源和氮源分别 为葡萄糖和酵母膏。在此基础上采用 Plackett-Burman 设计实验确定初始培 养基中影响酶产量的主要因素分别是葡萄糖、酵母膏和培养基 pH，经过爬 坡实验和 Box-Behnken 设计对培养基组成进行响应面优化，最终确定培养 基主要因素的最佳组合为葡萄糖为 0.314g/100mL， 酵母膏为 0.308g/100mL， 培养基 pH 为 7.41。在该培养基条件下，酶产量提高了 78%。在单因素实 验的基础上， 对影响发酵的几个因素， 温度()、 接种量(%)、 装液量 （mL） 和初始 pH 进行四因素三水平正交试验确定 Z4 产酶的最佳发酵条件。结果 表明温度对产酶的影响最大，其次是 pH、接种量和装液量。四个因素的最 佳组合为培养温度 28， 起始 pH7， 装液量 60mL,接种量 6%。 在该条件下， 酶产量提高了 79.6%。 在培养基优化过程中，还发现 Z4 菌株几丁质酶合成存在多态现象，因 此有必要对其几丁质酶基因的表达调控进行深入研究。此外将几丁质酶基 因克隆到高效表达载体中使其进行异源表达，这样可以去除供体菌自身对 产几丁质酶的代谢控制，也可以提高酶产量。为此根据 GENE BANK 中唯 III 一的一个微杆菌属几丁质酶基因序列和几丁质酶催化域氨基酸保守序列设 计引物，对其保守区域进行 PCR 扩增和测序，获得催化域 192bp 的一段 DNA 序列。对该序列的生物信息学分析表明该基因为几丁质酶基因，属于 糖苷水解酶 18 家族。该序列的获得为进一步克隆其整个基因创造了条件， 为进一步基因的异源表达和分子改造奠定了基础。 CDA高产菌株的筛选首先是通过变色圈法筛选出产酶能力较强的菌株， 再通过比较发酵液中的酶活，筛选出产酶能力最强的菌株，编号为F2-7-3。 根据菌体的形态观察和16S rDNA序列分析，确定该菌株属于放线菌门 （Actinobacteria）的红球菌属（Rhodococcus） 。酶学性质的研究表明该酶催化 反应的最适pH为7.0、最适温度为50。为提高几丁质脱乙酰基酶的产量， 对发酵培养基的组成和发酵条件进行了优化， 首先通过单因素实验确定产酶 培养基最佳碳源和氮源分别为蔗糖和酵母膏， 通过正交试验确定初始培养基 中对产酶影响最大的无机盐是KH2PO4。据此，以发酵液酶活为响应值，采 用BoxBehnken设计构建了培养基影响产酶的三个主要因素，蔗糖、酵母 膏和KH2PO4的响应曲面模型。 该模型预测三个因素的最佳组合为： 蔗糖7g/L； 酵母膏9.15g/L；KH2PO4 0.93mmol/L；响应值即发酵液酶活力预测值为 250.61U/mL。经方差分析和实验验证，该模型是准确可靠的。在单因素实 验中， 当酶活最大时对应的培养温度、 pH和装液量分别为30、 pH 7和40mL。 本论文工作为几丁质的酶法制备以及壳聚糖和几丁寡糖的酶法生产提 供了新型的产酶菌株和基础数据，这对酶法生产壳聚糖和几丁寡糖具有极 其重要的意义。 关键词关键词：几丁质，几丁质酶，几丁质脱乙酰基酶，菌株，筛选，优化 IV V STUDIES ON THE KEY TECHNOLOGIES TO PRODUCE CHITIN AND DERIVATIVES WITH HIGH VALUE BY ENZYMATIC METHODS ABSTRACT Chitin is the most abundant renewable natural resource like cellulose. Billions tons of chitin is produced each year in nature, whereas a large amount of chitinolytics is produced to decompose the same amount chitins. The fact that chitin is broadly produced and stablely existed in nature shows and prove that the chitinolytic enzymes is existed broadly too. The diversity of environment and species of microorganisms make it possible to find chitinolytic enzymes with different properties. Chitin, as well as chitosan, which is deacetylse chitin, and their oligosaccharides forms has been studied and applied in many fields, such as food technology, phamorcology, material science, microbiology, tissue engineering, bionanotechnology and so on. So there comes the huge demand for this production. However, the conventional production of chitin, chitosan and their oligosaccharides involves the use of strong acids and base, which creates a disposal problem due to the large amounts of toxic waste that need further treatment and may pollute the environment. To overcome this problem, an alternative method using enzyme has been emerged, which can be in a considerable extent replace the non-environment friendly chemical process. Producing chitin and its related products by method of bioprocess is promising and interesting. Because of the milder producing conditions, and less environment pollution, the biological or enzymes method has become an ideal method replacing the chemical method, which is the focus. The recent studying focused on the methods of biological or enzymes methods which are ideal method with advantages such as mild producing conditions , less environmental pollutions. On account of these backgrounds, a novel process was applied to extract chitin from shrimps materials that pepsin and citrate was used to demineralization and deprotein. And then, novel strains with the high ability to produce chitinase or CDA was isolated from soils of Qinling Mountains that is VI different from marine environment used to isolated these strains. After that, the cultural medium and parameter of fermentation was optimized by one-factor experiment and response surface methodology (RSM). In this thesis, pepsin and citrate were applied for demineralization and deprotein during produce chitin. According to the ash content, the parameter of citrate solution, whose concentration and time were 12% and 13 hours, was decided by one factor experiment. Under this condition, the ash content of chitin was 1.5%, which is meet the requirment of industrial grade, and close to food grade. As for the deprotein studies, the optimal deproteinization conditions was studied by one factor experiment at first, and Kjeldahl method was used to determine the protein content. The data of one factor experiments indicates that the proper catalytic condition might be as follows, pH1.5, temperature 35, enzyme dose 500U/g, and the process time 4 hours, take the cost of production into account. On the basis of these results, response surface methodology (RSM) was used to study the optimal process conditions, the data indicates that the optimal condition occurs at 700 U/g, 36.09, and 5 hours, and the maximum deproteinization rate approaches 8.47%. The strain with significant ability is the foundation and the key of fermentation industrial, that is one of the most important work in the field of decompose chitin by microorganisms. In this study, soils in different envirement were collected from Qinling Mountains, and the strains with high ablity to produce chitinase or CDA screened after enrichment culture. Applying the isolation medium that colid chitin was the sole carbon source, 20 strains were selected according to the scale of clarity zone around the colony in medium. These strains were filtered further by fermentation, and the chitinase activity in supernatant was determined by DNA method. The first three strains with high ablitity producing chitnase were Z4, F9，and D5-23. The strain Z4 was the best one whose enzyme activity in supernantant reaches to 2.3 U/mL. Morphology and sequencing of the 16S rDNA information indicated that the strain was microbacterium sp. The chitinase production of Z4 was markedly enhanced by statistical optimization of medium composition and culture conditions. The single factor experiment shows that the glucose and yeast extract is the optimal source of carbon and nitrogen. The effect of glucose, yeast extract and initial pH VII on chitinase production was studied with method of Plackett-Burman design, and was then further optimized with the method of steepest ascent and RSM. Data show that 3.38g/L glucose, 3.24g/L yeast extract and an initial pH of 7.5 were optimum for the production of chitnase. According to the results of single factor experiment, the optimum technological parameters were ascertained by orthogonal experiment which is culture temperature 28, initial pH of 7，the volume of liquid 60mL, inoculum size 6%. Besides the optimizing the medium and fermentation parameter, the method of molecular biology were also used to study the property of the related genes because clone and express the chitinase gene in other microorganism is another useful method to enhance the production of chitinase. For the purpose of clone the chitinase gene of Z4, several PCR primers were designed according the sole chitinase gene from microbacterium genus and conserved amino acid sequence of chitinase. The genome DNA was extracted by kits and used as template for PCR amplification. The production was sequenced and analyzed by bioinformation methods. The results show that the sequence was part of chitnase gene, and belong to chitinase of GH 18 familys. In order to get new strains with powerful ability to produce CDA, 28 strains were isolated and screened out from the soil samples by method of color reaction in plate medium. And then, the paranitroacetanilide was used as the substrate to analyze the enzyme activity. The strain F2-7-3 was screened out from these strains for the highest CDA activity, which can reach more than 250U/mL. The morphological properties and 16SrDNA sequencing were studied and data suggested that the isolated strain belonged to the evolution branch of Rhodococcus. The enzyme activity was studied further, data shows that the optimum temperature was 50, the optimum pH was 7. The component of medium and parameters during fermentation were optimized respectively in order to improve the production efficient of CDA. According the results of single factor experiment and orthodox experiment, orthogonal experiment implies that the KH2PO4 was a most significant influence factor than other salts. Hence, the maximum chitinase activity of 250.61U/mL was obtained by using the optimized medium that contents of the three most important components, sucrose, yeast extract and KH2PO4 were 7g/L, 9.15g/L VIII and 0.93mmol/L respectively. Subsequencetly, the fermentation conditions of F2-7-3 were optimized by method of the single factor analysis to improve the fermentation process, the optimum conditions obtained were: temperature 30, pH 7 and liquid volume 40mL in 250Ml flask. New strains with excellent ability to produce enzymes and associated date were offered in the present studies, which are essential to produce chitin/chitosan and their oligosaccharide by enzymes, a kind of friendly method to environment. KEY WORDS: Chitin, chitinase, Chitin Deacetylase (CDA), strain, screen, optimum I 目目 录录 摘 要 . I ABSTRACT . V 1 综述 . 1 1.1 几丁质及其衍生物的研究进展 . 1 1.1.1 几丁质简介 . 1 1.1.2 几丁质来源 . 1 1.1.3 几丁质结构 . 1 1.1.4 几丁质的生物合成 . 2 1.1.5 基因工程菌株生产几丁质寡糖 . 2 1.1.6 几丁质及其衍生物的应用 . 3 1.1.7 几丁质的生物降解 . 7 1.1.8 几丁质、壳聚糖及其寡糖的制备 . 8 1.1.9 总结和展望 . 10 1.2 几丁质酶的研究进展 . 10 1.2.1 简介 . 10 1.2.2 微生物几丁质酶的来源和分类 . 11 1.2.3 微生物产几丁质酶特点 . 14 1.2.4 几丁质酶的酶学性质 . 14 1.2.5 微生物几丁质酶的分子生物学特性 . 15 1.2.6 微生物几丁质酶的应用 . 16 1.3 几丁质脱乙酰酶(CDA)的研究进展 . 18 1.3.1 产 CDA 的微生物 . 18 1.3.2 CDA 的理化性质 . 19 1.3.3 CDA 的催化机理 . 21 1.3.4 CDA 的酶活力测定方法 . 22 1.4 本论文的研究目的及设想 . 23 2 几丁质的酶法制备工艺研究 . 25 2.1 引言 . 25 2.2 材料与方法 . 25 2.2.1 主要试剂与仪器 . 25 2.2.2 实验方法 . 25 II 2.3 结果与讨论 . 26 2.3.1 柠檬酸脱钙 . 26 2.3.2 蛋白酶脱蛋白研究 . 28 2.4 小结 . 31 3 新型几丁质酶高产菌株的筛选及鉴定 . 32 3.1 引言 . 32 3.2 材料与方法 . 32 3.2.1 主要试剂与仪器 . 32 3.2.2 实验方法 . 33 3.3 结果与讨论 . 35 3.3.1 筛选 . 35 3.3.2 菌种鉴定 . 36 3.4 小结 . 37 4 几丁质酶高产菌株 Z4 的培养基优化 . 38 4.1 引言 . 38 4.2 材料与方法 . 38 4.2.1 主要试剂与仪器 . 38 4.2.2 实验方法 . 39 4.3 结果与讨论 . 40 4.3.1 生长曲线的测定 . 40 4.3.2 培养基诱导物种类的确定 . 40 4.3.3 培养基碳源种类的确定 . 41 4.3.4 培养基氮源种类的确定 . 42 4.3.5 Plackett-Burman 实验筛选主要因素 . 42 4.3.6 爬坡试验 . 44 4.3.7 响应面法优化发酵培养基 . 44 4.4 小结 . 48 5 几丁质酶高产菌株 Z4 的发酵条件研究 . 49 5.1 引言 . 49 5.2 材料与方法 . 49 5.2.1 主要试剂与仪器 . 49 5.2.2 实验方法 . 50 5.3 结果与讨论 . 51 III 5.3.1 单因素实验 . 51 5.3.2 正交试验 . 53 5.4 小结 . 55 6 菌株 Z4 几丁质酶基因部分序列的克隆及生物信息学分析 . 56 6.1 引言 . 56 6.2 材料与方法 . 56 6.2.1 实验材料 . 56 6.2.2 实验方法 . 56 6.3 结果与讨论 . 57 6.3.1 几丁质酶基因的扩增 . 57 6.3.2 测序及翻译结果 . 58 6.4 小结 . 60 7 几丁质脱乙酰基酶高产菌株的筛选鉴定 . 61 7.1 引言 . 61 7.2 材料与方法 . 61 7.2.1 实验材料 . 61 7.2.2 实验方法 . 62 7.3 结果与讨论 . 65 7.3.1 产酶菌株的筛选 . 65 7.3.2