Enhancing Protease and Amylase Activities in Bacillus licheniformis XS-4 for Traditional Soy Sauce Fermentation Using ARTP Mutagenesis

Abstract

This study was conducted to increase the enzymatic activity of Bacillus licheniformis XS-4, which was isolated from the traditional fermented mash of Xianshi soy sauce. The mutation was induced by atmospheric and room-temperature plasma (ARTP), and a mutant strain, mut80, was obtained. mut80 exhibited significant increases in protease and amylase activity by 90.54% and 143.10%, respectively, and the enhanced enzymatic activities were stably maintained after 20 consecutive incubations. Re-sequencing analysis of mut80 revealed that the mutation sites were located in 1518447(AT-T) and 4253106(G-A) in its genome, which was involved in the metabolic pathways of amino acids. The expression of the protease synthetic gene (aprX) increased 1.54 times, while that of the amylase gene (amyA) increased 11.26 times, as confirmed via RT-qPCR. Using ARTP mutagenesis, the present study proposes a highly efficient microbial resource with enhanced protease and amylase activity provided by B. licheniformis, which can potentially be used to improve the efficiency of traditional soy sauce fermentation.

Recent progress of atmospheric and room‐temperature plasma as a new and promising mutagenesis technology

“…In addition, chemical reactive species (RONS) produced by ARTP can also modulate the molecular structure of the enzyme, resulting in an increase in enzyme activity. 88,89 Zhang et al, 90 mutagenized Bacillus licheniformis using ARTP and obtained a mutant strain with significantly increased amylase and protease activities.…”

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.…”

Regulating Pathways of Bacillus pumilus Adamalysin-like Metalloendopeptidase Expression

“…Gene expression can be assessed directly through RT-qPCR, which is considered the most reliable and informative method [ 4 , 5 ]. However, gene expression can also be determined indirectly by evaluating the activity level of its product [ 6 , 7 ].…”