Cloning and expression of class I chitinases in Hami melon after Penicillium infection

Jiang, Fang; Shui, Bo; Tang, Fengxian; Shan, Chunhui

doi:10.4238/gmr16019085

Cited by 2 publications

(1 citation statement)

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“…The open reading frame (ORF) and encoded amino acid sequence of CHI were analyzed using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) followed by translation of the nucleotide sequence using DNAMAN software. Domain structure was analyzed at the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) and the three-dimensional structure of CHI was predicted using SWISS-MODEL (https://swissmodel.expasy.org/) (Jiang et al, 2017). A phylogenetic neighbor-joining tree was constructed using MEGA 5.0 software, and ClustalW was used for sequence alignment (Liu et al, 2018;Noby et al, 2018).…”

Section: Bioinformatic Analysesmentioning

confidence: 99%

Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

Cong

Shen

et al. 2020

PeerJ

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Background Chitinases 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. Methods Primers 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. Results The 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. 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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Section: Bioinformatic Analysesmentioning

confidence: 99%