PURIFICATION AND CHARACTERIZATION OF AMYLASE PRODUCED FROM PROBIOTIC LACTOBACILLUS PLANTARUM CS FOR INDUSTRIAL APPLICATIONS

Pakistan Journal of Biotechnology Pub Date : 2024-04-14 DOI:10.34016/pjbt.2024.21.02.890

U. Nwachukwu, U. George-Okafor, K. Mba-Omeje, Amara Chioma Ezeme-Nwafor, Ifeoma Agatha Onah, Ifeanyi Jude Victor Egbuji

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Abstract

Previous studies have demonstrated that probiotic Lactobacillus plantarum CS was able to generate an appreciable amount of extracellular amylase, hence the need to purify and characterize it. The aim of the study was to purify and characterize crude amylase from probiotic Lactobacillus plantarum CS for its industrial applications Three purification steps including ammonium sulphate precipitation, ion exchange chromatography on carboxymethyl sephadex and gel filtration on Sephadex G-75 were utilized. The homogeneity of the purified enzyme was confirmed using sodium deodocyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified amylase was characterized on different parameters including substrates hydrolyses, pH and temperature activity and stability profiles. The general purification elution profile revealed two different peaks of amylase activities with outstanding one having a molecular weight of 59.7kDa. Its purification fold was 4.0 with specific activity of 16.44U/mg protein and enzyme yield of 3%. Temperature optimal activity and stability was at 400C and 7.5 for pH activity and stability. Mangenese (Mn2+) (135.17%), tween 80 (128.30%) and some food condiments garlic, thyme, ginger, and tumeric) significantly (p> 0.05) enhanced amylase activity (≥262.40%). However, selenium (Se4+) and hydrogen peroxide (H2O2) were observed to have greatest inhibiting effect (≥30.9%) on the enzyme. Substrate hydrolysis profiles showed that the amylase hydrolyzed all the test starchy substrates with the highest hydrolytic potential on indigenous sweet potato starch (Km value/ Vmax of 1.33mg/ml/ 7.89ml). The rate of hydrolysis of other test substrates had yam> rice>cassava>corn with km values ≤ 4.0mg/ml and Vmax ≤ 25ml. The obtained results gave an insight that amylase produced from Lactobacillus plantarum CS met with the possessed properties suitable for any industrial application especially in food

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纯化和表征植物益生菌 Cs 生产的淀粉酶，用于工业应用

以往的研究表明，益生菌植物乳杆菌 CS 能够产生相当数量的胞外淀粉酶，因此有必要对其进行纯化和鉴定。本研究的目的是纯化植物乳杆菌 CS 的粗淀粉酶并确定其特性，以便将其用于工业用途。本研究采用了三个纯化步骤，包括硫酸铵沉淀、羧甲基sephadex 离子交换色谱和 Sephadex G-75 凝胶过滤。纯化酶的均一性是通过硫酸脱十二烷基钠聚丙烯酰胺凝胶电泳（SDS-PAGE）确认的。对纯化的淀粉酶进行了不同参数的表征，包括底物水解、pH 值和温度活性以及稳定性曲线。一般纯化洗脱图显示了两个不同的淀粉酶活性峰，其中分子量最大的一个为 59.7kDa。其纯化倍数为 4.0，比活性为 16.44U/mg，酶产率为 3%。最佳活性和稳定性温度为 400℃，最佳 pH 值为 7.5。锰（Mn2+）（135.17%）、吐温 80（128.30%）和一些食物调味品大蒜、百里香、生姜和苤蓝能显著（p> 0.05）提高淀粉酶的活性（≥262.40%）。然而，硒（Se4+）和过氧化氢（H2O2）对淀粉酶的抑制作用最大（≥30.9%）。底物水解曲线显示，淀粉酶可水解所有测试的淀粉底物，其中对本地甘薯淀粉的水解潜力最大（Km 值/Vmax 为 1.33 毫克/毫升/ 7.89 毫升）。其他测试基质的水解率依次为山药>大米>木薯>玉米，Km 值≤ 4.0 毫克/毫升，Vmax ≤ 25 毫升。所得结果表明，植物乳杆菌 CS 生产的淀粉酶具有适合任何工业应用（尤其是食品工业）的特性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Pakistan Journal of Biotechnology

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