Abstract：
In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg-1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg-1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg-1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5\'-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme\'s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL-1 and 1580.3 ± 183.7 μmol mg-1 protein min-1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.
摘要：
在生物技术领域，利用农业工业废物生产高价值产品，如微生物生物量和酶，具有重要意义。本研究的目的是利用乳清作为有用的生长培养基，从卡瓦氏芽孢杆菌K1菌株中生产重组α-淀粉酶。纯化的六组氨酸标记的α-淀粉酶表现出显著的同质性，具有1069.2Umg-1的比活性。该酶在55°C和pH6.5时显示出其峰值活性，即使在55°C下孵育3小时后仍保留约70％的活性。它的分子量,通过SDS-PAGE测定，大约69kDa。α-淀粉酶对小麦淀粉表现出高活性（1648.8±16.8Umg-1），而对环糊精和直链淀粉表现出相对较低的活性（≤200.2±16.2Umg-1）。它表现出对盐的特殊耐受性，耐受浓度高达2.5M。有趣的是，金属离子和洗涤剂，如十二烷基硫酸钠（SDS），Triton100、Triton40和Tween80,5,5'-二硫代-双-[2-硝基苯甲酸(DNTB),β-巯基乙醇(ME),和二硫苏糖醇（DTT）对酶的活性没有明显的抑制作用，CaCl2（2mM）的存在甚至导致重组酶的轻微激活（1.4倍）。米氏常数（Km）和最大反应速率（Vmax），以可溶性淀粉为底物，产生的值分别为1.2±0.19mgmL-1和1580.3±183.7μmolmg-1蛋白min-1。值得注意的是,通过用β-葡萄糖苷酶处理酸乳清24h，获得了生物质和重组α-淀粉酶生产的最有利条件。
