Isolation, Molecular Characterization, Optimization and Purification of Amylase Enzyme from Locally Isolated Bacillus Species in Different Regions of Munshiganj, Bangladesh

Md. Fuadh-Al-Kabir, Julia Ferdouse, A.N.M. Hamidul Kabir
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Abstract

Amylase is a kind of enzyme that facilitates the breakdown of carbohydrates in the body. This enzyme has many applications in different industries. Generally, in pharmaceutical sector it is used for the treatment of pancreatic disorder, pancreatic enzyme replacement therapy (PERT) and as a digestive aid. In this study, the bacterial strain was isolated from soil samples collected from potato dumpsites in different areas of Munshiganj, Bangladesh. After subculturing on the nutrient agar plate, 31 colonies were obtained, of which 9 isolates were found as amylase producer on starch agar medium. Of these, 2 isolates (MC-04 and MC-15) were selected based on the starch hydrolysis clear zone ratio. The isolate MC-04 showed crude enzyme activity of 2.82 IU/ml and specific enzyme activity of 3.42 IU/mg, and isolate MC-15 showed crude enzyme activity of 3.16 IU/ml and specific enzyme activity of 3.77 IU/mg. The best isolate, MC-15, was then identified by morphology, biochemical and molecular characterization and confirmed by 16S-rRNA gene sequencing. After 16S-rRNA sequencing, the isolate (MC-15) was identified as Bacillus subtilis. This amylase production of this strain had also been optimized under certain conditions such as different incubation periods, pH, temperatures and different carbon sources. We found that the best incubation period was 48 h, the optimum pH-7.0, the optimum temperature at 40°C and 2% starch was considered as the best source of carbon. Finally, the crude amylase enzyme was purified by precipitation with ammonium sulfate, dialysis, and single-step gel filtration chromatography. The enzymatic activity of the purified amylase was found to be 8.91 IU/ml, that was 2.82-fold greater enzymatic activity than the raw enzyme. The experiments confirmed that Bacillus subtilis may be a good source of amylase enzyme for industrial application in Bangladesh. Dhaka Univ. J. Pharm. Sci. 22(2): 147-154, 2023 (December)
孟加拉 Munshiganj 不同地区当地分离的芽孢杆菌淀粉酶的分离、分子特征、优化和纯化
淀粉酶是一种促进体内碳水化合物分解的酶。这种酶在不同行业有许多应用。一般来说,在制药领域,它被用于治疗胰腺疾病、胰酶替代疗法(PERT)和消化辅助剂。在这项研究中,细菌菌株是从孟加拉国 Munshiganj 不同地区的马铃薯倾倒地采集的土壤样本中分离出来的。在营养琼脂平板上进行亚培养后,获得了 31 个菌落,其中 9 个分离菌株在淀粉琼脂培养基上可产生淀粉酶。根据淀粉水解透明区比率,选出了其中的 2 个分离物(MC-04 和 MC-15)。MC-04 分离物的粗酶活为 2.82 IU/ml,特异酶活为 3.42 IU/mg;MC-15 分离物的粗酶活为 3.16 IU/ml,特异酶活为 3.77 IU/mg。随后,通过形态学、生化和分子特征鉴定,并通过 16S-rRNA 基因测序确认了最佳分离物 MC-15。经过 16S-rRNA 测序,该分离株(MC-15)被确定为枯草芽孢杆菌。在不同的培养期、pH 值、温度和不同碳源等条件下,对该菌株的淀粉酶产量也进行了优化。我们发现,最佳培养期为 48 小时,最佳 pH 值为 7.0,最佳温度为 40°C,2% 的淀粉被认为是最佳碳源。最后,通过硫酸铵沉淀、透析和单步凝胶过滤色谱法纯化了粗淀粉酶。纯化后的淀粉酶的酶活为 8.91 IU/ml,是原酶的 2.82 倍。实验证实,枯草芽孢杆菌可能是孟加拉国工业应用淀粉酶的良好来源。Dhaka Univ.22(2):147-154,2023 年(12 月)
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