【作者】 姜绪林；

【导师】 张星元；

【作者基本信息】 江南大学 ， 发酵工程， 2005， 硕士

【摘要】 纤维素是植物细胞壁的主要成分,广泛而大量地存在于自然界中,是地球上最为丰富的可再生的生物聚合物之一。从高效和环保的角度出发,纤维素被彻底分解而无污染的一条有效的途径便是利用纤维素酶的水解作用。纤维素酶的来源非常广泛,但除个别的研究目的外,利用微生物以外的生物生产纤维素酶缺乏现实意义,而采用微生物发酵法是最方便的方法。 本论文以提高绿色木霉产纤维素酶的固态发酵水平为主要的研究目的,首先对固态发酵培养物中的丝状真菌生物量的测定方法进行了研究,确定了细胞壁中麦角固醇的提取工艺,建立了麦角固醇含量与菌丝体质量之间的线性关系。 目前,有关纤维素酶的研究很多,但是各个单位的酶活测定方法和对酶活单位的定义各有不同,这给酶活大小的相互比较带来了困难。为此,我们在实验初期重新定义了酶活单位,并对纤维素酶的活性测定方法进行了优化,确定了适用于本菌株产生的纤维素酶活性测定的一种较优方案。 为了充分的提取发酵结束后固体曲中的纤维素酶,研究了纤维素酶的浸提条件,发现一定质量的酶曲按15mL/g干曲加入蒸馏水,在40℃,80r/min下提取1.0h,其提取效果最好。由此得到的酶液,用饱和度为35%的硫酸铵溶液进行盐析,可以去除部分杂蛋白,并且酶活回收率在90%左右。在此基础上研究了粗酶液中纤维素酶对热和对酸碱度的稳定性,并确定了其最适的作用温度和作用pH值分别为60℃和pH5.0。还对常见的八种无机阳离子对纤维素酶活性的影响进行了研究。 最后,通过对发酵工艺的营养条件和培养条件的优化,确立以一套较优的固态发酵工艺:250mL三角瓶,装固体料5.3g(其中已被利用两次的稻草粉5.0、豆饼粉0.3g),水料质量比2.2(其中,无机营养液的成分为:蔗糖2%、硫酸铵3%、乳糖0.6%、硫酸镁0.25%、吐温802%、自然pH),接种浓度为3×10~5个/mL的孢子悬浮液1mL,发酵温度前期控制为32℃,后期为30℃,静置培养6d。发酵工艺优化前后CMC酶活和FPA酶活分别提高了80.8%和88.9%。更多还原

【Abstract】 Cellulose, the main composition of the vegetal cell wall, exists widely in the nature and is the most abundant reproducible bio-polymer in the earth. In view of the high efficiency and the environment protection, the degradation catalysed with cellulase is the most effective way to hydrolyze cellulose thoroughly and causes no pollution. Cellulase can be extensively obtained, but except for some particular research, its production with other life-form but microorganism is unpractical, and the microbial fermentation is considered as the most convenient way.This research was mainly aimed at the improvement of the cellulase production with solid fermentation by Trichoderma viride. Firstly, in order to detect the biomass of the filamentous fungi in the solid culture medium, a preferable process for extracting ergosterol from the fungi cell wall was determined. Based on this, the linear relation between ergosterol and biomass was established.The studies about cellulase are widely performed, and the measurement and the definition of the cellulase activity unit in one laboratory differ with the others, which brings some difficulty to the comparison of the cellulase activity. Therefor, in the beginning of this research the activity unit was redefined and the method for measuring the activity of cellulase produced by this strain was optimized.In order to extract the cellulase from the solid culture medium completely, the extraction process was studied, and finally determined as following: adding 15 mL distilled water to every one gram dried koji, then extracting cellulase at 40℃ with a rotate speed of 80 r/min for about an hour. The liquid obtained in this way was salt-outed with 35% saturated （NR₄）₂SO₄ solution, thus, about 90% of the cellulase activity was conserved and some of the other proteins were removed. Then the cellulase stabilities in this crude enzymatic liquid were studied, including thermal stability and acid stability. The optimum temperature and pH for the hydrolysis were also determined as 60℃ and 5.0 respectively. In addition, the effect of eight general inorganic positive ions on the cellulase activity was also observed.Finally, on the basis of the optimization of the nutrition and the circumstance condition for the fermentation, a set of preferable solid fermentation process was established as following: 5.3 g solid culture medium, including 5.0 g straw powder which had already been used twice and 0.3 g bean cake powder, was put into a 250 mL flask, then add in some nutritious solution, containing 2% sucrose, 3% （NH₄）₂SO₄, 0.6% lactose, 0.25% MgSO₄ and 2% Tween-80, with a qualitative ratio which is 2.2 to the solid material, then inoculating with 1 mL spore suspension whose concentration is 3×10⁵/mL and cultivated at 32 ℃ in the prophase and then adjusted to30*C for 6 days totally. Compared to the former process, after the process optimization the activities of CMCase and FPAse were increased by 80.8% and 88.9% respectively.更多还原