Manipulation of amylase reaction to improve the reducing sugars production

Abstract

An Anoxybacillus strain SK3-4 was previously isolated from Perak Sungai Klah hot spring. The a-amylase gene fragment from Anoxybacillus sp. denoted as ASKA was cloned into pET-22b(+) and transformed into Escherichia coli BL21 (DE3). However, the reactivity and productivity of this amylase is underexplored. The main objective of this project is to optimize the reducing sugars production using Response Surface Methodology (RSM). The ASKA substrate specificity was determined using soluble starch and nine different commercial starches: corn, tapioca, wheat, potato, rice, sago, rye, green peas and glutinous rice starch. Sago starch was found to be the best substrate with highest reducing sugars production. Variable parameters such as reaction temperature, sago starch and ASKA concentration were screened using one-factor-at-a-time (OFAT) approach before they were optimized through two-level full factorial design and central composite rotatable design (CCRD). Statistical analysis showed that all the three parameters were significant factors in 23 full factorial design before further optimized the reducing sugars production with CCRD. The final optimized parameters using CCRD was capable to produce 7.97 g/L reducing sugars with 2.64 % (w/v) sago starch and 0.375 unit ASKA under 66.9 ºC reaction temperature. The hydrolysis products were determined using High Performance Liquid Chromatography (HPLC). Maltose was the major hydrolysis product and no glucose production was detected. As a conclusion, applying experimental designs method was able to improve the efficiency of reducing sugars production for 87.09 % compared with the reference reaction condition with maltose as the major end product.