Abstract
BackgroundChitinases are enzymes which degrade β-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance.MethodsPrimers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out.ResultsThe exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg−1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0–10.0 and 25–45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and β-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products.ConclusionsThis is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.
Highlights
Chitin is composed of linear chains of poly-β-1,4-N-acetylglucosamine, and is found in the exoskeletons of arthropods and fungal cell walls
We isolated a marine bacterium capable of producing chitinase from a sample of shrimp-pond bottom mud from mangroves in the Maowei Sea in China, which we identified to be Paenibacillus chitinolyticus strain UMBR 0002
We report the cloning, heterologous expression and purification of a chitinase (CHI) from P. chitinolyticus strain UMBR 0002 for the first time
Summary
Chitin is composed of linear chains of poly-β-1,4-N-acetylglucosamine, and is found in the exoskeletons of arthropods and fungal cell walls. Degradation of chitin by chitinases is controllable and low cost and can produce high yields in mild conditions with minimal environmental impacts (Deng et al, 2019; Kidibule et al, 2018; Salas-Ovilla et al, 2019). The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Results: The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc) as products. Conclusions: This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste
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