Yield Enhancement of Recombinant α-Amylases in Bacillus amyloliquefaciens by ARTP Mutagenesis-Screening and Medium Optimization

Abstract

α-Amylase is the most extensively applied enzyme in industry. There is an urgent need for improvement on the yield of α-amylases currently. Herein, a strategy which combined Atmospheric and Room Temperature Plasma (ARTP) mutagenesis tool for construction of mutant library of Bacillus amyloliquefaciens with a 24-well plates screening technique was adopted to improve the yield of recombinant Bacillus amyloliquefaciens α-amylases (BAA). A mutant strain named B. amyloliquefaciens ZN mut-7# was obtained, and the activity of BAA produced by this mutant strain was 86.92% higher than that of the original strain. B. amyloliquefaciens ZN mut-7# has an unchanged BAA gene and genetic stability. This successful application proved that ARTP can be applied to the genetically engineering strains that contain recombinant plasmid. Furthermore, response surface methodology offers an achievable and efficient strategy to optimize the composition of medium used to generate BAA in B. amyloliquefaciens ZN mut-7#. A 1.28-fold increase had been obtained compared to the production of non-optimized fermentation medium. This study demonstrates that ARTP mutagenesis and medium optimization are efficient and feasible methods for increasing recombinant enzyme production in the genetically engineering strains.

Optimisation and production of alpha amylase from thermophilic Bacillus spp. and its application in food waste biodegradation

“…Similar results were reported by Swain et al (2006) in which the optimum activity of α-amylase extracted from B. subtilis CM3 was found at pH 5.0-9.0 and 50-70 C. While Awasthi et al (2018a,b) reported four isolates of Bacillus with maximum amylase activity at pH 5.3-8.2 and 60 C. The level of amylase production was found to depend on the temperature effects on the growth of the organism. The temperature range of 35-80 C has earlier been reported to favour the growth α-amylase -producing bacteria (Bajpai and Bajpai, 1989;Burhan et al, 2003;Ibrahim et al, 2013;Asad et al, 2014;Xu et al, 2019). However, temperatures above 65 C were found to negatively affect the activity of enzymes while a further increase in temperature caused a decline in the activity of α-amylase (Figure 5) possibly due to enzyme denaturation which led to inhabitation (Anto et al, 2006).…”

Amylolytic microorganisms from diverse tropical environments: Isolation, identification, and amylase production

“…The reasons for lower/varied specific activity could be enzyme production and assay conditions, such as pH and temperature, were not optimized in this study. Amylase-producing enzymes have optimum growth and enzyme-producing conditions (pH and temperature) [24,25,30].…”

Application of Atmospheric and Room-Temperature Plasma (ARTP) to Microbial Breeding

“…The alkaline phosphatase activity of the mutant strain S-52 was determined to be 12,110.6 U/L, which is 4.67 times greater than that of the original strain. ARTP has also been employed in the production of other enzymes, such as protease [ 34 , 35 ], alginate lyase [ 36 ], α-amylase [ 37 ], and alkaline protease [ 38 ]. The mutant strains exhibited significantly higher enzyme production capacity compared to the original strain, demonstrating the effectiveness of ARTP in improving enzyme yield.…”