Abstract
Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with β-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.
Highlights
According to an estimate, approximately 6–8 million tons of crab, shrimp, and lobster shell waste is produced annually around the world
Our results show that a newly discovered chitinase from M. xanthus could be a promising candidate for the eco-friendly bioconversion of chitin waste to its highly valued products
The full-length open reading frame (ORF) encoding a putative chitinase candidate in M. xanthus consisting of 1731 bp base pair was successfully amplified and ligated into the expression vector pET30a (Figure S1)
Summary
Approximately 6–8 million tons of crab, shrimp, and lobster shell waste is produced annually around the world. Most of it is directly dumped into the sea or landfill [1], used for composting, and to produce fertilizers and animal feed [2]. It is well known that in addition to protein and calcium carbonate, shells contain chitin, the second most available polysaccharide after cellulose. The content of chitin reaches 15–40% dry weight of the shells of crustaceans [5]. The potential value of such shells in the chemical or biotechnology industry field is normally neglected
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