Optimization of phenylalanine production by Bacillus subtilis isolated from dark soil using response surface methodology

Abstract

One of the most appealing technologies for the industrial manufacture of amino acids like phenylalanine is microbial fermentation. Phenylalanine has a wide range of industrial uses and is a crucial raw material in the production of aspartame, a low-calorie sweetener that is thought to be at least 150 times sweeter than sucrose and has a sizable global market. Thus, the goal of this study was to identify microorganisms that can produce phenylalanine and to set up the conditions for maximal output. The bacteria were isolated from the soil, identified biochemically and molecularly, and subsequently tested for the ability to produce phenylalanine. Phenylalanine production was initially optimized using a combination of the One-Factor-at-a-Time (OFAT) approach later Response Surface Methodology (RSM) with a Central Composite Design (CCD)." a higher output of 2.89±0.01 mg/mL of phenylalanine was observed by OFAT. At pH 6.5, temperature 40 ^oC, agitation rate 125 rpm, and incubation duration 58 hours, RSM produced the highest yield of phenylalanine (3.63 mg/mL), with a significant quadratic model at a p-value less than 0.05 and a several significant effects were identified, including linear and quadratic effects of pH, temperature, and incubation time, along with pH-temperature and temperature-agitation rate interactions. The functional groups (amino group, carboxylic group, and aromatic group) in the manufactured phenylalanine were identical to those in the phenylalanine that was purchased from a store, according to Fourier Transform Infrared Spectroscopy study. Thin layer chromatography (TLC) analysis of the generated phenylalanine revealed a Retardation factor value (0.6) identical to that of phenylalanine obtained from commercial sources.  Using high performance liquid chromatography (HPLC), it was possible to detect bacterially synthesized phenylalanine and measure its concentration (2.8 ± 0.05 mg/mL). According to the study, the isolated bacteria may be able to produce phenylalanine in large quantities.