Kahar Ummirul Mukminin, Sani Mohd Helmi, Chan Kok-Gan, Goh Kian Mau
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai 81310, Johor, Malaysia.
Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia.
Molecules. 2016 Sep 9;21(9):1196. doi: 10.3390/molecules21091196.
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
嗜热栖热放线菌SK3-4（ASKA）的α-淀粉酶是一种热稳定酶，可从淀粉中产生高水平的麦芽糖。在早期研究中对具有更高表达和纯化效率的截短型ASKA（TASKA）变体进行了表征。在本研究中，通过共价连接将TASKA固定在三种环氧化物（ReliZyme EP403/M、Immobead IB-150P和Immobead IB-150A）和一种氨基环氧化物（ReliZyme HFA403/M）活化载体上。分析了影响固定化的几个参数，包括pH值、温度以及每克载体添加的酶量（毫克）。研究了载体表面性质、孔径和间隔臂（官能团）长度对生物催化剂性能的影响。游离和固定化的TASKA在pH 6.0-9.0时稳定，在pH 8.0时具有活性。该酶在60℃时表现出最佳活性和相当的稳定性。固定化的TASKA在重复使用5-12个循环后保留了其初始活性的50%。在淀粉和直链淀粉降解时，只有固定在ReliZyme HFA403/M上的TASKA具有与游离酶相当的水解能力。据我们所知，这是关于嗜热栖热放线菌属α-淀粉酶固定化研究的首次报道，也是首次报道使用ReliZyme和Immobeads作为载体固定α-淀粉酶。
