| 高产碱性纤维素酶细菌的筛选鉴定及其酶学特性与发酵条件研究 |
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| 引用本文： | 马振刚,熊亮,张真,罗堿.高产碱性纤维素酶细菌的筛选鉴定及其酶学特性与发酵条件研究[J].南方农业学报,2021,52(3):722-731. |
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| 作者姓名： | 马振刚 熊亮 张真 罗堿 |
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| 作者单位： | 重庆师范大学生命科学学院, 重庆 401331 |
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| 基金项目： | 国家自然科学基金项目（31770160）；重庆市教委科学技术研究计划项目（KJQN201800524） |
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| 摘 要： | 【目的】从自然环境中分离筛选高产纤维素酶的菌株，开展碱性纤维素酶的酶学特性分析，为该菌株及所产纤维素酶的综合开发利用打下基础。【方法】采用羧甲基纤维素钠（CMCNa）平板筛选法筛选纤维素酶高产菌株，利用生理生化分析结合分子生物学法对菌株进行鉴定，并通过3， 5-二硝基水杨酸（DNS）法研究其活性特征与发酵条件。【结果】在长期覆盖枯树叶的土壤中分离获得1株高产碱性纤维素酶的菌株，经鉴定该菌株为蜡样芽孢杆菌（Bacillus cereus），名称为B. cereus strain CQNUX 3-1。酶活性分析显示该菌株胞外分泌液具有内切葡聚糖酶、外切葡聚糖酶及β-葡萄糖苷酶的活性，其酶活力分别达107.7、33.1和155.6 U/mL。酶学特征分析表明3种酶组分均具有较好的耐碱和一定的耐高温能力。其中，内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶的最佳反应温度分别为70、60和40℃；最佳反应pH分别为8.0、9.0和9.0；Fe3+能增加3种酶的酶活力，而β-葡萄糖苷酶具有较好的EDTA、尿素和Cu2+耐受性。发酵条件对菌株产酶的分析结果表明，该菌株发酵温度在37℃较适宜；发酵第4 d时的酶活力达最大值；该菌株能在碱性发酵环境下生长并产酶，在初始pH为7.0时发酵酶活力最高。【结论】筛选获得的纤维素酶高产菌株B. cereus strain CQNUX 3-1所生产的纤维素酶具有较高的反应温度适用性和较强的碱耐受性，菌株发酵产酶温度适中，且有较宽的发酵pH适用范围，可作为碱性纤维素酶生产资源菌株，具有应用于纤维素酶制剂制备与生产、纤维素资源综合利用等领域的潜力。
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| 关 键 词： | 碱性纤维素酶产生菌 筛选鉴定 酶学特性 发酵条件 蜡样芽孢杆菌 |
| 收稿时间： | 2020-11-13 |
Screening and identification of a strain with high yield of alkaline cellulase and its enzyme characterizations and fermentation conditions |
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| MA Zhen-gang,XIONG Liang,ZHANG Zhen,LUO Jian.Screening and identification of a strain with high yield of alkaline cellulase and its enzyme characterizations and fermentation conditions[J].Journal of Southern Agriculture,2021,52(3):722-731. |
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| Authors: | MA Zhen-gang XIONG Liang ZHANG Zhen LUO Jian |
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| Affiliation: | College of Life Sciences, Chongqing Normal University, Chongqing 401331, China |
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| Abstract: | 【Objective】Strains with high yield of cellulase were isolated from the natural environment, and the enzymatic characteristics of screened alkaline cellulase were analyzed which could lay a foundation for the comprehensive development and utilization of the cellulase produced by this strain.【Method】The carboxymethyl cellulose sodium(CMCNa) plate screening method was used to isolate and screen high yield cellulose-producing strains and then the isolated strains were identified by physiological and biochemical analysis and molecular biology method. The characteristics of enzyme activity and fermentation conditions were studied by 3, 5-dinitrosalicylic acid(DNS) method.【Result】Ahigh alkaline cellulase producing strain was isolated from the soil samples which were long-term covered by rotting leaves and this strain was identified and named as Bacillus cereus strain CQNUX 3-1. The results of enzyme activity tests showed that strain CQNUX 3-1 exhibited the activity of endoglucanase, exoglucanase and β-glucosidase and were 107.7, 33.1 and 155.6 U/mL, respectively. Analysis of enzymatic characterization showed that the three enzymes had better alkaline resistance and certain high temperature performance. The optimum reaction temperature of endoglucanase, exoglucanase and β-glucosidase were 70, 60 and 40℃, respectively. The best reaction pH were 8.0, 9.0 and 9.0 respectively. Fe3+ could increase the enzyme activity of three enzymes, while β-glucosidase showed better tolerance to EDTA, urea and Cu2+. The effect of fermentation conditions on the enzyme production showed that the optimum fermentation temperature of the strain was 37℃and the enzyme activity could reach to the maximum after four days fermentation. Finally, the bacteria could grow and produce enzymes under alkaline fermentation environment. Enzyme activity was the best when the initial pH was 7.0.【Conclusion】A strain with high alkaline cellulase producing is selected. The cellulase produced by B. cereus strain CQNUX 3-1 has high reaction temperature applicability and strong alkali resistance. The strain fermentation enzyme has moderate temperature and has a wide range of fermentation pH. This strain can be used as a alkaline cellulase production resource strain, with the potential for the preparation and production of cellulase preparations and the comprehensive utilization of cellulose resources. |
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