高效纤维素酶葡曲霉UCSC324的分离及其粗纤维素动力学性质的测定

S. Mohanappriya, R. Kapilan
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引用次数: 1

摘要

提高催化效率和热稳定性的纤维素水解酶的生物工程技术在商业化过程中具有重要意义。本文介绍了高效产纤维素酶真菌的分离及粗纤维素酶动力学性质的测定。在牛粪、热大米水、高压灭菌水和腐烂椰子木中分离的真菌菌株中,刚果红试验测定羧甲基纤维素钠盐琼脂板上的清除率,结果表明,在腐烂椰子木上生长的菌株纤维素酶产量较高。对从椰子木中分离得到的3株真菌进行形态学和分子分析,通过扩增ITS5.8SrDNA序列、PCR扩增和多重序列比对,鉴定为黑曲霉FL17、米曲霉CBS108.24和角曲霉UCSC324。由于目前还没有关于unguis Aspergillus UCSC324生产纤维素酶的报道,我们对该真菌菌株的纤维素酶动力学特性进行了研究。发酵培养基含(gL-1) 2.0g纤维素;3.0g羧甲基纤维素;0.3g硫酸铵和100mL蒸馏水在温度20±1oC, pH7.0,转速100rpm下作用5天。粗纤维素酶在5分钟内表现为零级动力学。在20℃~ 75℃(pH 7.0)温度范围内测定纤维素酶的活性,酶活性的最佳温度为50℃。当培养基pH由2.0变为8.0,温度保持在50℃,纤维素底物浓度为1g/100mL时,pH为5.0时纤维素酶活性最高。在pH 5.0和50℃条件下,采用Lineweaver-Burk法测定了Michaelis常数和纤维素酶对可溶性纤维素的Vmax分别为were4.545×10-2 moldm-3和26.66 mmml -2min -1。粗酶在pH 5.0和50℃条件下至少稳定90分钟。该酶在中酸性条件下具有较强的活性,在50℃条件下具有较好的稳定性,是纤维素酶工业应用的良好候选酶。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Isolation of efficient cellulase producing Aspergillus unguis UCSC324 and determination of the kinetic properties of its crude cellulose
Bioengineering of cellulolytic enzymes with enhanced catalytic efficiency and thermostability is important in the commercialization processes. This study describes the isolation of efficient cellulase producing fungi and determination of the kinetic properties of the crude cellulase. Among the fungal strains isolated from cow dung, hot rice water, water used in autoclave and decaying coconut wood, the strains growing on decaying coconut wood was selected for this study because of the higher amount of cellulase production measured by the rate of zone of clearance on the Carboxymethylcellulose (CMC)sodium salt agar plates by Congo red test. The three isolated fungal strains isolated from coconut wood were identified and confirmed as Aspergillus niger FL17, Aspergillus oryzae CBS108.24 and Aspergillus unguis UCSC324 based on the morphological studies and molecular analysis done by amplifying the ITS5.8SrDNA sequence, PCR amplification and multiple sequence alignment. Since there had been no reports recorded about the production of cellulase from Aspergillus unguis UCSC324, kinetic properties of the cellulase from this fungal strain were studied. Fermentation medium contained (gL-1) 2.0g cellulose; 3.0g carboxymethyl cellulose; 0.3g ammonium sulphate and 100mL of distilled water was used at an optimal conditions of temperature 20±1oC, pH7.0 for 5 days at 100rpm.Crude cellulase showed zero order kinetics for 5 minutes. When the activity of cellulase was measured at different temperatures ranging from 20°C to 75°Cat pH 7.0, the optimum temperature for the enzyme activity was 50°C. When the pH of the media was changed from 2.0 to 8.0, while temperature was kept at 50°C with 1g/100mL cellulose substrate, highest cellulase activity was observed at pH 5.0. Michaelis constant and the Vmax of the cellulase enzyme to soluble cellulose by Lineweaver-Burk Plot were4.545×10-2 moldm-3and 26.66 mgml-2mins-1respectively at pH 5.0 and 50°C. The crude enzyme was stable for at least 90 minutes at pH 5.0 and at 50°C. Since the cellulase enzyme from Aspergillus unguis was active in moderately acidic pH and showed better stability at 50°C, it could be a good candidate for the cellulase dependent industrial applications.
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