Optimization for high level expression of cold and pH tolerant amylase in a newly isolated Pedobacter sp. through Response Surface Methodology

Razie Ghazi-Birjandi, Bahar Shahnavaz, Maryam Mahjoubin-Tehran
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Abstract

Amylase is one of the most widely used enzymes in the industry. Cold environments are the most ubiquitous environments in the world that have been occupied by cold tolerant microorganisms. The enzymes of these microorganisms have a wide range of applications in various areas of biotechnology. The aim of this study was to isolate cold-active amylase producing bacteria. A total of 64 cold-tolerant bacteria producing amylase were isolated from Binaloud Mountain soil, Iran. An isolate (Pedobacter sp. BTR84) registered under accession number KM459538 with the highest enzyme productivity was selected for production optimization. The production of amylase was evaluated via One-factor-at-a-timemethod and RSM (Response Surface Methodology). The enzyme production was optimized at 20°C, pH 9, starch 2% (w/v), and inoculation level 3% (v/v) by One-factor-at-a-timemethod. Then, in order to investigate the interaction between these variables and determine the final optimal conditions, optimization was carried out through the response surface methodology (RSM). Four variables were evaluated at three levels using the Box-Behnken design. Starch concentration and inoculation level variables had a significant effect on amylase production (p<0.05). The final optimum conditions for amylase production in the isolates were temperature 25°C, pH 7, starch concentration 2.5% (w/v), and inoculation level 3% (v/v). The current study suggests that Psychrotrophic local bacteria are capable of producing extracellular hydrolytic enzymes that have a good potential to be applied in biotechnological industries.
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响应面法优化新分离的耐冷耐pH淀粉酶的高表达
淀粉酶是工业上应用最广泛的酶之一。寒冷环境是世界上最普遍存在的耐寒微生物所占据的环境。这些微生物的酶在生物技术的各个领域有着广泛的应用。本研究的目的是分离产生冷活性淀粉酶的细菌。从伊朗Binaloud山地土壤中分离到64株产生淀粉酶的耐寒细菌。选择酶产率最高的分离物(Pedobacter sp. BTR84)进行生产优化,注册号为KM459538。通过单因子法和响应面法评价淀粉酶的产量。采用单因素单次法,在20℃、pH 9、淀粉2% (w/v)、接种量3% (v/v)条件下优化产酶。然后,为了研究这些变量之间的相互作用,确定最终的最优条件,通过响应面法(RSM)进行优化。使用Box-Behnken设计在三个水平上评估四个变量。淀粉浓度和接种水平对淀粉酶产量有显著影响(p<0.05)。菌株产生淀粉酶的最佳条件为温度25℃,pH 7,淀粉浓度2.5% (w/v),接种量3% (v/v)。目前的研究表明,嗜冷性局部细菌能够生产胞外水解酶,在生物技术工业中具有很好的应用潜力。
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