1、106个/mL,培24h,PEC酶活力和Endo-PG酶活力分别为973.47U/mL和165.08U/mL。通过对粗酶液作用pH和温度的研究,得到PEC和Endo-PG为酸性果胶酶,最适pH为4.5,PEC和Endo-PG最适温度分别为45 C和55 C。半连续发酵试验结果表明,在第5批次出现最高PEC酶活力和Endo-PG酶活力,分别为1253.95U/mL和181.94U/mL,分别比首次提高29.8%和18.3%。然后,对烟梗浸液发酵产果胶酶进行研究。采用单因素和正交试验法优化培养基,结果表明影响产果胶酶的因素依次为:烟梗硫酸铵Tween80。烟梗10%,硫酸铵1.5%,ZnSO40.
2、6 mmol/L,Tween80 0.05%,K2HPO4 0.2%,KH2PO4 0.2%。烟梗10%,硫酸铵1.5%,ZnSO40.6 mmol/L,Tween80 0.05%,K2HPO4 0.2%,KH2PO4 0.2%,发酵初始pH 5.0,初始孢子浓度0.5106,30C,装液量50mL,转速170r/min,培养48h,PEC酶活力和Endo-PG酶活力最高分别为361.27U/mL和49.22U/mL。烟梗浸液发酵96h,出现纤维素酶活力高峰,滤纸酶活力和羧甲基纤维素钠酶活力分别为33.25U/mL和83.13U/mL。半连续发酵试验结果表明,在第2批次出现最高PEC酶活力和E
3、ndo-PG酶活力,分别为430.83U/mL和51.73U/mL,PEC酶活力比首批次提高约22.8%。关键词:米根霉,果胶酶,聚半乳糖醛酸内切酶,固定化,半连续化发酵ABSTRCTNatural cellulose is one of the renewable resources, such as orange peel and tobacco stem, which are made of cellulose, half fiber, lignin and pectin. The intertwined combination among them leads to form a ti
4、ght and complex structure, which is hard for microorganisms to digest. Hence, the production of lactic acid or ethanol through using natural cellulose by microorganisms is faced with problems. Rhizopus oryzae has the ability to produce pectinolytic enzymes to degrade pectin.The remove of pectin can
5、make separation of fiber, fiber and lignin easier. There is a growing market demand of pectinolytic enzymes for the fact that pectinolytic enzymes are widely used in the filed of food, textile, pharmaceutical, paper making, biological technology and so on. Rhizopus oryzae, which owns the ability to
6、degrade pectin and is rich in comercial value, is used to produce pectinolytic enzymes in this research. The immobilized Rhizopus oryzae with matrix composed of asterisk-configuration fibrous matrices in a honeycomb-shaped, the production of pectinolytic enzymes were carried out from citrus pectin a
7、nd tobacco stem pectin. Pectinolytic enzymes (PEC) activity was determined by DNS assay and endopolygalacturonase (Endo-PG) activity was measured by viscosity method. Fistly, the pure pectin was selected as the sbustrate for fermentation. Then, the coparison of enzymes production between immobilized
8、 cells and free cells was made. After that, optimization of culture medium and growth conditions was investigated through single factor variable analysis and so on. Also, some properties of the enzyme were under reasearch. Finally, optimization of culture medium made from tobacco stem was investigat
9、ed through single factor variable analysis and orthogonal experiment. Optimization of growth conditions was investigated through single factor variable analysis. Six batches of semi-continuous fermentation were carried out in shake flasks.First of all, the study of pure pectin was carried out. Immob
10、ilized cells had a higher production of PEC over free cells. Immobilized cells had a max production in 48 hours, which saved 24 hours compared with free cells and improved production by 115%. Results of single factor and orthogonal experimental with immobilized showed the order of factors influencin
11、g the production of pectinase was: pectin Tween80 ZnSO4 (NH4)2SO4.The suitable culture media was composed of pectin2.5%, (NH4)2SO4 1.5%, ZnSO40.6 mmol/L ,Tween80 0.15%, K2HPO4 0.4%,KH2PO4 0.4%,rotation speed 190 r/min, liquid volume 50 ml/250 ml, temperature 30C, initial pH 5.0, initial spore concen
12、tration 0.75106 /mL. After 24 hours, the PEC activity and Endo-PG activity were 973.47U/mL and 165.08U/mL separately. Effects of pH and temperature on crude enzymes fluid were carried out.PEC and Endo-PG might be acidic pectinolytic enzymes. The optimum pH was 4.5 for PEC and Endo-PG, optimum temper
13、ature was 45Cand 55C respectively. The highest PEC activity and Endo-PG activity were 1253.95U/mL and 181.94U/mL in the 5th batch, about 29.8% and 18.3% higher than the 1st batch. Secondly, optimization of culture medium made from tobacco stem was investigated through single factor variable analysis
14、 and orthogonal experiment. Results showed the order of factors influencing the production of pectinase was: tobacco stem (NH4)2SO4ZnSO4Tween80. The suitable culture media was composed of tobacco stem 10%, (NH4)2SO4 1.5%, ZnSO40.6 mmol/L,Tween80 0.05%, K2HPO4 0.2%,KH2PO4 0.2%,rotation speed 170 r/mi
15、n, liquid volume 50 ml/250 ml, temperature 30C, initial pH 5.0, initial spore concentration 0.50106 /mL. After 48 hours, the PEC activity and Endo-PG activity were 361.27U/mL and 49.22U/mL separately. After 96 hours, cellulose enzyme activity reached a peak with filter paper activity of 33.25U/mL an
16、d sodium carboxymethyl cellulose activity of 83.13U/mL.The highest PEC activity and Endo-PG activity were 430.83U/mL and 51.73U/mL in the 2nd batch. PEC activity was about 22.8% higher than the 1st batch.Keywords: Rhizopus oryzae, pectinolytic enzymes, endopolygalacturonase, immobilization, semi-con
17、tinuous fermentation目 录摘 要 IABSTRCT II1绪论 11.1问题的提出及研究意义 11.1.1问题的提出 11.1.2研究意义 21.2国内外研究现状 21.2.1果胶分布、化学结构和分类 21.2.2果胶酶分类和作用方式 31.2.3果胶酶生产菌种 41.2.4真菌果胶酶的表达调控 61.2.5 微生物果胶酶条件优化研究现状 71.2.6米根霉产果胶酶研究进展 71.2.7固定化细胞产果胶酶的进展 81.2.8果胶酶生产过程中的影响因素 91.2.9果胶酶的应用 111.2.10果胶质原料的研究 121.3本课题研究的目的及主要内容 121.3.1研究目的 1
18、21.3.2本课题研究的主要内容 121.3.3创新点 132.棉布载体固定化发酵产果胶酶的研究 142.1前言 142.2材料和方法 142.2.1菌种 142.2.2主要仪器和试剂 142.2.3培养基 152.2.4载体的制备 152.2.5发酵培养方法 152.2.6分析方法 162.3结果分析 182.3.1酶液稀释倍数的确定 182.3.2游离发酵与固定化发酵的比较 192.4讨论 192.4.1酶液稀释倍数对酶活力测定准确性的影响 192.4.2游离发酵与固定化发酵 192.5小结 193固定化发酵果胶酶纯果胶培养基的优化 203.1前言 203.2材料和方法 203.2.1菌种
19、 203.2.2主要仪器和试剂 203.2.3培养基 213.2.4发酵培养方法 213.2.5分析方法 213.3结果分析 223.3.1碳源种类的选择 223.3.2果胶浓度选择 233.3.3氮源种类选择 233.3.4硫酸铵浓度选择 243.3.5 Tween80对产酶的影响 253.3.6金属离子的对产酶的影响 253.3.7发酵培养基的正交实验 263.4讨论 273.4.1碳源种类及果胶浓度对产酶的影响 273.4.2氮源种类及硫酸铵对产酶的影响 283.4.3Tween80对产酶的影响 283.4.4金属离子对产酶的影响 283.4.5正交实验结果分析 293.5小结 294纯
20、果胶培养基半连续发酵果胶酶 304.1前言 304.2材料和方法 304.2.1菌种、主要仪器和试剂 304.2.2培养基 304.2.4发酵培养方法 314.2.5分析方法 314.3结果分析 314.3.1转速的选择 314.3.2装液量选择 324.3.3温度选择 324.3.4初始pH的选择 334.3.5初始孢子浓度的选择 334.3.6生长和产酶的时间曲线 344.3.7半连续发酵试验 354.3.8粗酶液部分酶学性质 364.4讨论 374.4.1转速和装液量对产酶的影响 374.4.2 pH对产酶的影响 374.4.3初始孢子浓度对产酶的影响 384.4.4产酶与pH变化的关系
21、 384.4.5半连续发酵的稳定性 384.4.6粗酶液部分酶学性质 384.5小结 395. 固定化发酵果胶酶烟梗浸液培养基的优化 405.1前言 405.2材料和方法 405.2.1试剂和设备 405.2.2烟梗浸液的制备 415.2.3培养基及发酵培养方法 415.2.4分析方法 415.3结果分析 415.3.1两种制备烟梗浸液方法的比较 415.3.2烟梗浓度的选择 425.3.3硫酸铵浓度的选择 425.3.4 Zn2+离子浓度的选择 435.3.5 Tween80浓度的选择 435.3.6发酵培养基的正交试验 445.4讨论 455.4.1制备烟梗浸液方法 455.4.2培养基的
22、优化 455.5小结 456. 烟梗浸液培养基半连续发酵果胶酶 476.1前言 476. 2材料和方法 476.2.1试剂和设备 476.2.2烟梗浸液的制备 476.2.3培养基及发酵培养方法 476.2.4分析方法 486.3结果分析 496.3.1初始pH的选择 496.3.2初始孢子浓度的选择 496.3.3生长和产酶的时间曲线 506.3.4半连续发酵试验 516.3.5最优发酵条件下纤维素酶活力 516.4讨论 526.4.1发酵条件的优化 526.4.2生长和产酶的时间曲线 526.4.3半连续发酵的稳定性 536.4.4发酵过程中纤维素酶活力的变化 536.5小结 537.结论
23、与展望 547.1结论 547.2对后续工作的展望 55致 谢 56参考文献 57附 录 621绪论1.1问题的提出及研究意义1.1.1问题的提出天然纤维素原料如橘皮、烟梗、稻草、玉米秸秆等是被废弃的资源,同时由于其内部纤维素与果胶之间紧密作用造成利用生物技术转化天然纤维素原料时的困难。天然纤维素原料在进行微生物转化过程中,一般需要先进行半纤维素和木质素等预处理过程,然后进行纤维素酶解和微生物发酵产乙醇、丙醇、丁醇、柠檬酸和乳酸等。预处理过程的好坏很大程度上决定了酶解的难易程度,如酶结合性和催化水解率1。果胶质的存在对半纤维、木质素去除的有一定程度的影响,主要表现在增加处理过程中溶液的粘度和溶
24、剂的使用量。米根霉在微生物转化纤维素的众多研究中有着重要的地位。米根霉作为一种公认的安全菌,能产生广泛的代谢物,如酶类(如果胶酶等),有机酸(如乳酸等)以及生物乙醇等,有着极大商业价值2。果胶酶在食品、纺织、医药、造纸、环境、生物技术、饲料等领域都有广泛应用3,这就使得果胶酶的需求量逐年递增。果胶酶生产量占全部工业酶制剂的10%左右4,其中在食品酶的销售额中约占到了25%5。固定化米根霉的形式可视为特殊的生物活性结构单元,它可以将精制原料如淀粉、葡萄糖等或是天然纤维素原料如玉米秸秆、稻草等转换为乳酸、乙醇等,但还未应用于降解果胶质产酶的研究。国内外在米根霉利用果胶质这一领域尚存在诸多空白,为数
25、不多的研究6-12,主要着力在固态发酵产果胶酶酶性质上的研究,如聚半乳糖醛酸酶和聚半乳糖醛酸裂解酶等的性质,未涉及固定化米根霉降解果胶的研究,对产酶培养基和培养条件的研究也不足,因此降解果胶产果胶酶的能力不高。细胞固定化与细胞游离培养相比有许多优势,如细胞密度的增加,细胞产率的提高,细胞分离更加方便从而有利于半连续和连续发酵13-14。真菌细胞的固定可以通过吸附于聚合物载体或者包埋于天然聚合物(海藻酸盐凝胶和合成凝胶)。凝胶颗粒包埋细胞易阻碍物质传输,影响发酵能力,还需要耗费时力制备大量凝胶颗粒。棉布载体固定化细胞的方式由于将细胞吸附于棉布表面,同时金属网状支撑骨架利于物质传输,因此菌体形态和
26、发酵效果好。此外,棉布载体还具有重复使用性好和工业组装方便等特点,具有工业化生产的潜力15。据此,本文设想将棉布载体固定化米根霉这一生物活性单元引入到降解果胶质产酶的研究中,优化生物活性单元产酶的培养基和培养条件,并进行半连续试验,考察载体的重复使用性和稳定性。1.1.2研究意义本文试图将一种研究较少但有产果胶酶能力且富有商业价值的安全菌种-米根霉,结合以棉布载体固定化细胞的技术,用于降解果胶质的研究,有助于天然纤维素生物转换为下游产品如乳酸和乙醇等,同时收获果胶酶粗酶液。1.2国内外研究现状1.2.1果胶分布、化学结构和分类果胶,最早由Bracennot在胡萝卜中提取得到的16。果胶广泛存在
27、于高等植物的根、茎、叶和果实中,水果皮如柑橘、柠檬、柚子等含有约30%的果胶,商品天然果胶多用酸从这些果皮或果渣中提取制得。 果胶,分子式为(C6H10O6)n,相对分子质量5万-30万。果胶在高等植物初生壁和胞间层中存在,在初生壁中与木质纤维的微纤丝以及某些伸展蛋白相互交联作用下,使得细胞组织结构坚固, 维持细胞固有的形态。它是以-1,4糖苷键键合D-半乳糖醛酸形成的多糖主链,同时与带有鼠李糖、半乳糖和阿拉伯糖等中性糖侧链共价键合的聚合物。果胶骨架基本单位是D-半乳糖醛酸,同时还含有如甲醇、乙酸和阿魏酸等非糖成分,其中D-半乳糖醛酸上的羧基可被甲基不同程度的脂化。一般可将果胶的结构分为光滑区
28、和须状区, 主要由三个结构域组成,即HGA、RG-I和RG-II,其中RG-II常为二聚体形式(如图1.1 所示)。果胶有多分子、多分散、多结构,高级空间构象的特点 17。果胶质可分三类,即原果胶(protopectin)、果胶(pectin)和果胶酸(pectic acid)。1944年,果胶术语制定委员会对果胶物质作出明确的定义,具体如表1.1所示18 。表1.1果胶物质的定义Tab.1.1 The definition of pectic substances分类定义溶解性果胶酸(pectic acid, pectate)完全未甲酯化的聚半乳糖醛酸链,由D-半乳糖醛酸脱水缩合,以-1,4
29、糖苷键连接形成的直链状聚合物微溶于水(pectin, pectinate)羧基不同程度甲酯化和中和的聚半乳糖醛链,分为低酯果胶(LM)和高酯果胶(HM)可溶于水原果胶(protopectin)存在于未成熟果蔬的细胞壁中,与纤维和半纤维结合的甲酯化聚半乳糖醛酸链不溶于水图1.1 果胶的结构简图Fig. 1.1 Schematic representation of the primary structure of pectin1.2.2果胶酶分类和作用方式果胶酶通常分为原果胶酶、酯酶和解聚酶三种类型:原果胶酶将不溶于水的原果胶降解为高度聚合的可溶性果胶;酯酶通过对甲基的去除,促使果胶酯水解;解聚酶打断果胶质
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