【作者】 缑敬轩；

【导师】 董文宾；

【作者基本信息】 陕西科技大学 ， 应用化学， 2012， 博士

【摘要】 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（EC3.2.1.14）、几丁质脱乙酰基酶(CDA; EC3.5.1.41)和壳聚糖酶(EC3.2.1.99)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700U/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2.3U/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Box—Behnken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；KH2PO40.93mmol/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℃、pH7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。更多还原

【Abstract】 Chitin is the most abundant renewable natural resource like cellulose.Billions tons of chitin is produced each year in nature, whereas a large amountof chitinolytics is produced to decompose the same amount chitins. The fact thatchitin is broadly produced and stablely existed in nature shows and prove thatthe chitinolytic enzymes is existed broadly too. The diversity of environmentand species of microorganisms make it possible to find chitinolytic enzymeswith different properties.Chitin, as well as chitosan, which is deacetylse chitin, and theiroligosaccharides forms has been studied and applied in many fields, such asfood technology, phamorcology, material science, microbiology, tissueengineering, bionanotechnology and so on. So there comes the huge demand forthis production. However, the conventional production of chitin, chitosan and their oligosaccharides involves the use of strong acids and base, which creates adisposal problem due to the large amounts of toxic waste that need furthertreatment and may pollute the environment. To overcome this problem, analternative method using enzyme has been emerged, which can be in aconsiderable extent replace the non-environment friendly chemical process.Producing chitin and its related products by method of bioprocess ispromising and interesting. Because of the milder producing conditions, and lessenvironment pollution, the biological or enzymes method has become an idealmethod replacing the chemical method, which is the focus. The recent studyingfocused on the methods of biological or enzymes methods which are idealmethod with advantages such as mild producing conditions, less environmentalpollutions. On account of these backgrounds, a novel process was applied toextract chitin from shrimps materials that pepsin and citrate was used todemineralization and deprotein. And then, novel strains with the high ability toproduce chitinase or CDA was isolated from soils of Qinling Mountains that isdifferent from marine environment used to isolated these strains. After that, the cultural medium and parameter of fermentation was optimized by one-factorexperiment and response surface methodology (RSM).In this thesis, pepsin and citrate were applied for demineralization anddeprotein during produce chitin. According to the ash content, the parameter ofcitrate solution, whose concentration and time were12%and13hours, wasdecided by one factor experiment. Under this condition, the ash content of chitinwas1.5%, which is meet the requirment of industrial grade, and close to foodgrade. As for the deprotein studies, the optimal deproteinization conditions wasstudied by one factor experiment at first, and Kjeldahl method was used todetermine the protein content. The data of one factor experiments indicates thatthe proper catalytic condition might be as follows, pH1.5, temperature35℃,enzyme dose500U/g, and the process time4hours, take the cost of productioninto account. On the basis of these results, response surface methodology (RSM)was used to study the optimal process conditions, the data indicates that theoptimal condition occurs at700U/g,36.09℃, and5hours, and the maximumdeproteinization rate approaches8.47%. The strain with significant ability is the foundation and the key offermentation industrial, that is one of the most important work in the field ofdecompose chitin by microorganisms. In this study, soils in different envirementwere collected from Qinling Mountains, and the strains with high ablity toproduce chitinase or CDA screened after enrichment culture. Applying theisolation medium that colid chitin was the sole carbon source,20strains wereselected according to the scale of clarity zone around the colony in medium.These strains were filtered further by fermentation, and the chitinase activity insupernatant was determined by DNA method. The first three strains with highablitity producing chitnase were Z4, F9，and D5-23. The strain Z4was the bestone whose enzyme activity in supernantant reaches to2.3U/mL. Morphologyand sequencing of the16S rDNA information indicated that the strain wasmicrobacterium sp. The chitinase production of Z4was markedly enhanced bystatistical optimization of medium composition and culture conditions. Thesingle factor experiment shows that the glucose and yeast extract is the optimalsource of carbon and nitrogen. The effect of glucose, yeast extract and initial pH 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 that3.38g/L glucose,3.24g/L yeast extract and an initial pH of7.5were optimum for the production of chitnase. According to the results of singlefactor experiment, the optimum technological parameters were ascertained byorthogonal experiment which is culture temperature28℃, initial pH of7，thevolume of liquid60mL, inoculum size6%.Besides the optimizing the medium and fermentation parameter, themethod of molecular biology were also used to study the property of the relatedgenes because clone and express the chitinase gene in other microorganism isanother useful method to enhance the production of chitinase. For the purposeof clone the chitinase gene of Z4, several PCR primers were designed accordingthe sole chitinase gene from microbacterium genus and conserved amino acidsequence of chitinase. The genome DNA was extracted by kits and used astemplate for PCR amplification. The production was sequenced and analyzed bybioinformation methods. The results show that the sequence was part of chitnase gene, and belong to chitinase of GH18familys.In order to get new strains with powerful ability to produce CDA,28strainswere isolated and screened out from the soil samples by method of color reactionin plate medium. And then, the paranitroacetanilide was used as the substrate toanalyze the enzyme activity. The strain F2-7-3was screened out from thesestrains for the highest CDA activity, which can reach more than250U/mL. Themorphological properties and16SrDNA sequencing were studied and datasuggested that the isolated strain belonged to the evolution branch ofRhodococcus. The enzyme activity was studied further, data shows that theoptimum temperature was50℃, the optimum pH was7.The component of medium and parameters during fermentation wereoptimized 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 KH2PO4was a most significant influencefactor than other salts. Hence, the maximum chitinase activity of250.61U/mLwas obtained by using the optimized medium that contents of the three most important components, sucrose, yeast extract and KH2PO4were7g/L,9.15g/Land0.93mmol/L respectively. Subsequencetly, the fermentation conditions ofF2-7-3were optimized by method of the single factor analysis to improve thefermentation process, the optimum conditions obtained were: temperature30℃,pH7and liquid volume40mL in250Ml flask.New strains with excellent ability to produce enzymes and associated datewere offered in the present studies, which are essential to producechitin/chitosan and their oligosaccharide by enzymes, a kind of friendly methodto environment.更多还原