Enzymatic conversion strategies of starch to maltooligosaccharides by maltogenic amylase and cyclodextrin glucanotransferase

Abstract

Recently, the application of emulsion liquid membrane (ELM) process as an alternative technology for solute separation is highlighted due to the simple operation of simultaneous extraction and stripping process. The most important aspects for a successful ELM process are liquid membrane formulation and emulsion stability. This study was carried out to investigate the liquid membrane formulation for the reduction of chromium (VI) to chromium (III) from electroplating wastewater using continuous ELM process (CELM). Liquid membrane system comprises of three liquid phases which are external (electroplating wastewater), organic liquid membrane and internal phase. Liquid membrane and internal phase were emulsified and dispersed into the external phase to be treated. The experimental work consisted of four major parts which were ELM component formulation, stability study of ELM in batch process, screening of parameters and optimization of chromium removal efficiency by response surface methodology (RSM) in continuous operation process and recovery of the chromium at optimum process conditions. The results show that the favourable conditions for liquid membrane formulation are 0.04 M TOMAC as a carrier, palm oil as a diluent and 0.1 M thiourea in 0.1 M sulfuric acid as a stripping agent. The best condition of stable water-in-oil (W/O) emulsion was obtained at 7000 rpm of homogenizer speed, 5% (w/v) Span 80 as surfactant and 1 minute of emulsifying time. Meanwhile, the most stable water-in-oil-in-water (W/O/W) emulsion obtained during the continuous process operation was at 350 rpm agitation speed, pH<5 of external phase and 1 to 5 of treat ratio. The optimization results by RSM show that 99% of chromium was extracted at 2.83 minutes of retention time, 342 rpm rotational speed and 1 to 5 of treat ratio. As a conclusion, about 81% of less-toxic chromium (III) has been recovered into the internal phase using 2.0 M thiourea in 2.0 M sulfuric acid as the stripping agent. The favourable process condition of the formulated membrane study was satisfactory and is suitable to treat wastewater as low as 20 ppm up to 200 ppm of chromium concentrations. This study reveals that CELM is a simple process and practical technology to remove chromium (VI) from industrial wastewater while solving the environmental problem simultaneously.In nature, Bacillus lehensis G1 utilizes extracellular cyclodextrin glucanotransferase (CGTase) to degrade starch into cyclodextrins (CDs). This is followed by hydrolysis of CDs by intracellular maltogenic amylase (MAG1) into glucose, maltose and maltooligosaccharides (MOS). The MOS are potential prebiotic for human consumption. In industries, amylases are used to produce MOS from starch. However, the conversion of starch directly to MOS using MAG1 has several limitations such as low specificity towards starch compared to ß-cyclodextrin (ß-CD) and low productivity of MOS. In order to overcome these drawbacks, two strategies involving optimization of reaction parameters using statistical method and synergism of enzyme mixture approach, were applied. In this study, the optimization of enzymatic reaction parameters for enhanced MOS production by MAG1 using soluble starch as a substrate was performed. In the first strategy, the effects of reaction parameters (enzyme loading, substrate loading, temperature, reaction time and pH) on MOS yield was investigated using one-factor-at-a-time (OFAT) method and 25-1 fractional factorial design. Based on the 25-1 fractional factorial design results, three parameters namely substrate loading, reaction time and pH were found to have a significant effect and was used in central composite design under response surface methodology (RSM). The MOS production was successfully optimized by the RSM. Under the optimized conditions (0.25 % (w / v) of substrate loading, 0.5 h of reaction time and pH 7.45) by RSM, the MOS yield was 107.29 mg / g of substrate which was 1.3-fold higher compared to the value after OFAT analysis which was only 84.87 mg / g of substrate. In the second strategy, the synergistic effect of MAG1 and CGTase for improving the MOS production process had also been studied using two different approaches which were asynchronous and synchronous methods. For the asynchronous method, the cyclization and hydrolysis reaction of CGTase and MAG1, were carried out in two separated steps respectively. Whereas, for the synchronous method, the two enzymes were added simultaneously and became a one-pot enzymatic reaction. The results from the studies conducted show the capability of the synchronous method was capable to convert the soluble starch (1.5 % (w / v)) into MOS with higher yield than the asynchronous method. The optimum conditions were obtained when MAG1 loading to CGTase loading in 3 U: 7 U ratio, with the reaction temperature of 40 ºC and pH 7.0. Based on these optimum conditions, the total yield of MOS attained was 307.86 mg / g of substrate after 2 h, which was 2.1-fold higher than the asynchronous method (146.78 mg / g) and 2.9-fold higher compared to the reaction of MAG1 alone. The used of CGTase and MAG1 synchronously enable the direct conversion of soluble starch to higher yield of MOS.