Fermentation optimization and amylase activity of endophytic Bacillus velezensis D1 isolated from corn seeds

Abstract

Aims: To acquire quality amylase adopted in practical applications, endophytic bacteria were identified as Bacillus velezensis strain D1 which was isolated from corn seeds. The fermentation conditions and amylase properties of the strain were investigated. Methods and Results: The strain D1 was identified via morphological, physiological and 16S rDNA phylogenetic analysis. The fermentation conditions of secreting amylase were optimized by single-factor and orthogonal experiments. The α-amylase gene was expressed in E. coli and purified by means of immobilized metal ion affinity chromatography (IMAC), upon which the enzyme activity of purified recombinant α-amylase was determined. The results outlined that (1) The strain D1 was identified as Bacillus velezensis. (2) The optimized fermentation conditions for maximum amylase yields included 44°C for 48 h at pH 7.5. (3) The enzyme had an optimal reaction temperature of 60°C with the highest activity at 50°C and tolerance to 4-h incubation at 70°C. (4) The enzyme was strong acid resistant and tolerated at pH 5.0–6.0 while the optimal pH was 8.0. (5) Besides, the amylase activity was elevated by the presence of Ca2+ and Cu2+. (6) The activity of purified recombinant amylase was 20.59 U/ml under optimal conditions, nearly seven times that of crude amylase preparations. Conclusions: The amylase produced by Bacillus velezensis D1 is strongly tolerant towards acid and high temperatures. Significance and Impact of the Study: Amylases with thermophilic and acid-resistant characteristics are useful for a wide range of applications in food, brewing, textile, starch, paper and deterrent industries. The enzyme from Bacillus velezensis D1 can be effectively used in different areas of industries.