High-Efficiency Secretion and Directed Evolution of Chitinase BcChiA1 in Bacillus subtilis for the Conversion of Chitinaceous Wastes Into Chitooligosaccharides

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.

Directed Evolution: Methodologies and Applications

“…Such approaches are beneficial for identifying hot spots as well as reducing library sizes. MD simulation-assisted directed evolution methods have been used to engineer proteins with improved catalytic activity, − enantioselectivity, − stability, specificity, and thermostability …”

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

“…Only three previous studies describe the directed evolution of chitinases, all of which involve low-throughput screening methods based on agar plate or MTP assays [ 19 , 20 , 21 ]. The natural substrate of ChiA is chitin or partially acetylated chitosan, because it has an absolute requirement for N -acetyl- d -glucosamine and cleaves glycosidic bonds at random internal sites immediately downstream of an N -acetyl- d -glucosamine unit [ 36 ].…”

“…Directed evolution has been applied to a variety of enzymes with industrial applications, such as glucose oxidase [ 9 , 10 ], P450 monooxygenases [ 11 ], cellulases [ 12 ], peroxidases [ 13 ], cellobiose dehydrogenase [ 14 ], transaminases [ 15 ], esterases [ 16 ], lipases [ 17 ], proteases [ 18 ], and many more. However, there are only a few examples of the directed evolution of chitinases [ 19 , 20 , 21 ].…”

Distribution and Biotechnological Applications of Chitinase: A Review

“…Use of a chitinase from Aspergillus niger (AnChiB) has been observed for bioconversion of fungal mycelial waste into N-acetyl-D-glucosamine [105]. Wang et al (2020) studied the high efficiency production and directed evolution of chitinase BcChiA1 in Bacillus subtilis and utilized it for bioconversion of chitinous wastes into chitooligomers [106].…”