摘要
【目的】筛选沼气发酵液中高产纤维素酶菌株,并对产酶条件进行优化,为沼气发酵利用和纤维素酶的工业化开发提供高效菌株。【方法】利用刚果红平板染色法初筛,纤维素酶活测定进行复筛,从沼气发酵池中分离产纤维素酶菌株,并结合形态学观察、生理生化特征和16S rDNA基因序列同源性分析进行鉴定。对菌株产酶培养基中碳源、氮源、硫酸镁和磷酸二氢钾添加量进行单因素优化,以滤纸酶活为指标,并在优化的基础上进行正交试验。对菌株接种量、培养时间、初始pH值、温度条件进行单因素优化,在单因素试验基础上采用响应面法Box-Behnken试验设计(BBD)优化纤维素降解菌产酶工艺的最佳工艺参数。【结果】初步确定该菌株发酵产酶培养基最佳条件为羧甲基纤维素钠含量0.75%、蛋白胨含量6%、硫酸镁添加量0.05%、磷酸二氢钾添加量0.2%;产纤维素酶最佳工艺条件为温度36℃,初始pH值为7.0,接种量4%,培养时间24 h,优化后其滤纸酶活达到98.49 U/mL,与优化前相比提高了约1.6倍。【结论】根据菌体的形态学观察、生理生化特征和16S rDNA基因序列同源性分析,表明该菌株为Bacillus amyloliquefaciens,本研究为沼气发酵利用和纤维素酶的工业化开发提供了高效菌株,建议进一步研究纤维素酶的生产利于其在工业水平上的应用。
【Objective】In this work,the authors screened the high-producing cellulase strains in the biogas fermentation broth,and optimized the enzyme production conditions,in order to provide efficient strains for the utilization of biogas fermentation and the industrial development of cellulase.【Method】By using Congo red plate staining method and cellulase activity determination,the primary screening and rescreening were conducted,respectively.Then the cellulase-producing strains were isolated from the biogas fermentation tanks and identified by morphological observation,physiological and biochemical characteristics and 16S rDNA gene sequence homology analysis.The single-factor optimization of the carbon source,nitrogen source,magnesium sulfate and potassium dihydrogen phosphate in the enzyme production medium was carried out,and by using the filter paper enzyme activity as the indicator,orthogonal experiments were carried out on the optimization basis.The single-factor optimization of strain inoculation amount,culture time,initial pH and temperature conditions was carried out.Based on the results,the response surface method Box-Behnken experimental design(BBD)was used to optimize the parameters of the enzyme production process of cellulose-degrading bacteria.【Result】The optimal fermentation medium for this strain was preliminarily determined,which contained 0.75%sodium carboxymethyl cellulose,6%peptone,0.05%magnesium sulfate,0.2%potassium dihydrogen phosphate.The optimal processing conditions for cellulose production were at a temperature of 36℃,and an initial pH of 7.0,an inoculum amount of 4%and a culturing time of 24 h.After the optimization,the filter paper enzyme activity reached 98.49 U/mL,which was about 1.6 times higher than that before optimization.【Conclusion】According to the morphological observation,physiological and biochemical characteristics and 16S rDNA gene sequence homology analysis,the selected strain was proved to be Bacillus amyloliquefaciens.This study provided a highefficiency str
作者
温冬灼
张智
魏罡
张晓彤
WEN Dongzhuo;ZHANG Zhi;WEI Gang;ZHANG Xiaotong(Key Laboratory of Forest Food Resources Utilization of Heilongjiang Province,Northeast Forestry University,Harbin 150040,Heilongjiang,China;College of Forestry,Northeast Forestry University,Harbin 150040,Heilongjiang,China)
出处
《中南林业科技大学学报》
CAS
CSCD
北大核心
2022年第2期169-180,共12页
Journal of Central South University of Forestry & Technology
基金
内蒙古自治区科技成果转化项目(CGZH2018179)。
关键词
芽孢杆菌
产酶条件优化
响应面
滤纸酶活
筛选
bacillus
optimization of enzyme production conditions
response surface
filter paper enzyme activity
screening
