Gene cloning and expression of the l-asparaginase from Bacillus cereus BDRD-ST26 in Bacillus subtilis WB600

Abstract

l-Asparaginase (ASN; EC 3.5.1.1) shows great commercial value because of its ability to reduce toxic levels of acrylamide in foods. To achieve high-efficiency production of l-asparaginase, an open reading frame of 978 bp encoding asparaginase (BcA) was amplified from Bacillus cereus BDRD-ST26, followed by its expression in Bacillus subtilis WB600, with the highest yield of 374.9 U/ml obtained using an amyE-signal peptide. A four-step purification protocol was used to purify BcA, resulting in a 15.1-fold increase in purification yield, with a specific activity of purified BcA at 550.8 U/mg and accompanied by detection of minimal l-glutaminase activity. Maximum BcA activity was detected at 50°C and pH 9.0 in 20mM Tris-HCl buffer, with a half-life at 50°C of 17.35min and a Km and kcat of 9.38mM and 63.6s-1, respectively. Compared with untreated potato strips, 72% acrylamide (2.35mg/kg) was removed from potato strips pretreated with BcA. These results indicated that this novel BcA variant represents a potential candidate for application in the food-processing industry.