Department of Biotechnology, Maulana Abul Kalam Azad University of Technology, West Bengal, Kalyani, India.
Department of Biotechnology, University of Engineering and Management, Kolkata, West Bengal, India.
Appl Biochem Biotechnol. 2023 Sep;195(9):5439-5457. doi: 10.1007/s12010-022-04043-y. Epub 2022 Jul 6.
The enzyme endoglucanase is responsible for the depolymerization of cellulose. This study focuses on characterization and purification of endoglucanase from Rhizopus oryzae MTCC 9642 through a simple size exclusion method and its effective application as an antibiofilm agent. Extracellular ß-1,4-endoglucanase, an enzyme that catalyzes the hydrolysis of carboxymethyl cellulose, was found to be synthesized by Rhizopus oryzae MTCC 9642. The enzyme was purified up to homogeneity simply by size exclusion process through ultrafiltration and gel chromatography. The molecular weight of purified enzyme protein was estimated to be 39.8 kDa and it showed the highest substrate affinity towards carboxymethyl-cellulose with K and V values of 0.833 mg ml and of 0.33 mmol glucose min mgprotein, respectively. The purified enzyme exhibited optimal activity at pH 6 with a broad stability range of pH 3-8. The most preferred temperature was 35 °C and 50% of activity could be retained after the thermal exposure at 40 °C for 25 min. The purified enzyme protein was inactivated by Cu, while the activity could be enhanced by the addition of exogenous thiols. Since biofilm is a challenge for health sector, with the aim of eradicating the biofilm, the purified endoglucanase was used to remove biofilm produced by two nosocomial bacteria. As predicted by in silico molecular docking interaction, the purified enzyme could effectively degrade biofilm architecture of bacterial strains S. aureus and P. aeruginosa by 76.52 ± 6.52% and 61.67 ± 8.76%, respectively. The properties of purified enzyme protein, as elucidated by in vitro and in silico characterization, may be favourable for its commercial applications as a potent antibiofilm agent.
内切葡聚糖酶负责纤维素的解聚。本研究通过简单的排阻方法对来自米根霉 MTCC 9642 的内切葡聚糖酶进行了表征和纯化，并将其有效应用于抗生物膜剂。发现产黄青霉 MTCC 9642 合成了一种能催化羧甲基纤维素水解的胞外 β-1,4-内切葡聚糖酶。该酶通过超滤和凝胶层析等简单的排阻过程可达到纯酶水平。纯化酶蛋白的分子量估计为 39.8 kDa，它对羧甲基纤维素表现出最高的底物亲和力，K 和 V 值分别为 0.833 mg ml 和 0.33 mmol 葡萄糖 min mgprotein。纯化酶在 pH 6 时表现出最佳活性，具有 3-8 pH 值的宽稳定性范围。最适温度为 35°C，在 40°C 下暴露 25 min 后，仍有 50%的活性保留。该纯化酶蛋白被 Cu 失活，而外源巯基的添加可增强其活性。由于生物膜是卫生保健部门面临的挑战，为了消除生物膜，将纯化的内切葡聚糖酶用于去除两种医院获得性细菌产生的生物膜。如通过计算机分子对接相互作用预测，纯化的酶可有效降解金黄色葡萄球菌和铜绿假单胞菌生物膜结构，分别为 76.52 ± 6.52%和 61.67 ± 8.76%。通过体外和计算机模拟表征阐明的纯化酶蛋白的特性，可能有利于其作为一种有效的抗生物膜剂的商业应用。
