Abstract
Limitations of enzyme production and activity pose a challenge for efficient degradation of chitinaceous wastes. To solve this problem, we engineered a system for high-yielding extracellular secretion of chitinase A1 from Bacillus circulans (BcChiA1) in B. subtilis. Furthermore, an innovative chitinase high-throughput screening method based on colloidal chitin stained with Remazol Brilliant Blue R (CC-RBB) was established and used to identify three mutants with improved chitinase activity: Y10A/R301A/E327A (Mu1), Y10A/D81A/E327A (Mu2), and F38A/K88A/R301A (Mu3). Their highest specific activity reached 1004.83 ± 0.87 U/mg, representing a 16.89-fold increase in activity compared to native BcChiA1. Additionally, we found that there is a synergistic effect between BcChiA1 and a lytic polysaccharide monooxygenase from Bacillus atrophaeus (BatLPMO10), which increased the chitin processing efficiency by 50% after combining the two enzymes. The yield of chitooligosaccharide (COS) production using the mutant Mu1 and BatLPMO10 reached 2885.25 ± 2.22 mg/L. Taken together, the results indicated that the CC-RBB high-throughput screening method is a useful tool for chitinase screening, and evolution of BcChiA1 in collaboration with BatLPMO10 has tremendous application potential in the biological treatment of chitinaceous wastes for COS production.
Highlights
Chitin is an insoluble, linear, high molecular weight polymer composed of β-1,4 linked N-acetylglucosamine (GlcNAc) units, and is one of the most ubiquitous polysaccharides in nature, second only to cellulose (Singh et al, 2017)
B. subtilis 1A751 was used as the expression host and plasmid pMATE was used for the expression of BcChiA1 and BatLPMO10
The results indicated that all signal peptides led to successful BcChiA1 secretion, but the noSP group performed best
Summary
Linear, high molecular weight polymer composed of β-1,4 linked N-acetylglucosamine (GlcNAc) units, and is one of the most ubiquitous polysaccharides in nature, second only to cellulose (Singh et al, 2017). It is the major structural component of a wide range of organisms, most notably fungi and arthropods, including species of shrimp and Chitinase Evolution and Chitin Biodegradation other seafood (Kumar et al, 2018). COSs have multiple promising applications in various fields such as food processing, biomedicine, agriculture, water purification, and cosmetics (Naveed et al, 2019)
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