摘 要

Abstract II

第一章 绪论 1

1.1引言 1

1.2纤维素 1

1.2.1纤维素的结构 2

1.2.2纤维素的性质 2

1.3纤维素酶 2

1.3.1纤维素酶的种类 3

1.3.2纤维素酶最适pH值及酶活的影响因子 3

1.3.31.3.3纤维素酶的应用 3

1.4降解纤维素的微生物种类 4

1.5酶促反应的热动力学研究 5

1.6本课题研究的意义、目的和技术路线 5

1.6.1研究的意义和目的 5

1.6.2研究拟采用的技术路线 6

第二章 实验材料及方法 7

2.1实验材料 7

2.1.1实验试剂 7

2.1.2实验仪器 8

2.2葡萄糖标准曲线的绘制 8

2.2.1所需试剂 8

2.3高温纤维素降解菌筛选与分离 10

2.3.1高温纤维素降解菌筛选培养基 10

2.3.2高温纤维素降解菌的活化 10

2.3.3涂布分离^[29] 11

2.3.4划线分离 12

2.4高温纤维素降解菌酶活检测 13

2.4.1实验原理及方法 13

2.4.2实验结果与分析 13

2.5高温纤维素降解菌产酶条件的优化 14

2.5.1碳源的优化 14

2.5.2氮源优化 15

2.5.3 pH值的优化 16

2.5.4 温度的优化 17

2.6对纤维素降解酶酶学特性的研究 18

2.6.1实验方法 18

2.6.2实验结果与分析 18

2.7实验小结 18

第三章 高温纤维素降解菌固体发酵技术的研究 20

3.1引言 20

3.2固体发酵模拟实验 20

3.2.1实验方法 20

3.2.2实验结果与分析 21

第四章 总结与展望 22

4.1 结论 22

4.2 存在的问题 22

4.3 未来展望 22

参考文献 23

致谢 25

摘 要

结构稳定的纤维素很难被分解利用，每年纤维素资源的浪费量巨大，如何高效分解利用纤维素是当前此研究领域的关键问题之一。化学法和物理法等传统处理方法均存在成本高、条件极端、易造成二次污染问题等缺点，而具有成本低、污染小等优点的微生物降解技术逐渐受到人们的重视。高温纤维素降解菌是微生物降解技术中主要的菌种之一，但目前研究中普遍存在酶活较低等问题。

本研究从某腐熟剂中分离出3株耐55℃高温的高效纤维素降解菌株A₄2、A₅3、A₆2，其最大纤维素酶活分别为14.52 U/ml、34.35 U/ml、22.64 U/ml。采用单因素实验进行生长条件的优化得出A₅3高温菌产纤维素酶的最适碳源、氮源、pH、温度分别为葡萄糖、蛋白胨、pH=7、55 ℃；通过酶学特性研究实验得到A₅3产纤维素酶的Km值为4.5×10^-3 g/ml，Vmax为0.055 umol/min·mL。使用3株菌株进行固体发酵实验，测得发酵产物中的纤维素酶活230.11 U/ml。

关键词：高温纤维素降解菌；纤维素酶；酶活；固体发酵

Abstract

The cellulose is difficult to be decomposed and utilized because of the structure is stable, and every year a huge amount of cellulose resources waste, so how to decompose and utilize cellulose efficiently is one of the key problems in this research field. The traditional methods such as chemical method and physical method have the disadvantages of high cost, extreme condition, and easy to be made into two pollution problems, however, it has been paid more and more attention to the technology of microbial degradation which has the advantages of low cost, small pollution and so on. High temperature cellulose degradation bacteria is one of the major microbial degradation technologies. But there are generally low enzyme activities in the study.

In this study, 3 strains of 55 degrees high temperature resistant efficient cellulose degrading strains A₄2、A₅3、A₆2 from a decomposition agent. The maximum cellulase activity were 14.52 U/ml、34.35 U/ml、22.64 U/ml. Growth conditions optimized thermophilic A₅3 cellulase production by the optimum carbon source, nitrogen source, pH and temperature were glucose, protein peptone, pH = 7, 55 ℃ by single factor experiment. In addition, the A₅3 production of cellulase by Km was 4.5×10^-3 g/ml and Vmax was 0.055 umol/min·mL obtained through the study of enzymatic properties. solid fermentation experiments were carried out to measure the activity of cellulase activity was 230.11 U/ml.

Key words: Thermophilic cellulose degrading bacteria; Cellulase; Enzyme activity; Solid fermentation