The broad-specificity chitinases: their origin, characterization, and potential application

Zhou, Jie; Chen, Jianhao; Xu, Ning; Zhang, Alei; Chen, Kequan; Xin, Fengxue; Zhang, Wenming; Ma, Jiangfeng; Fang, Yan; Jiang, Min; Dong, Weiliang

doi:10.1007/s00253-019-09718-x

Cited by 21 publications

(14 citation statements)

References 24 publications

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“…The former releases (GlcNAc) 2 from the non-reducing end of chitin chains. The latter acts on β-1, 4-glycosidic bonds at the end of products of low-molecular-mass GlcNAc oligomers and chitobiosidases to generate GlcNAc monomers (Hamid et al, 2013;Zhou et al, 2019). Bacterial chitinase offers wide environmental adaptability, fast expression, high thermal stability; furthermore, the enzyme can be genetically engineered (Le & Yang, 2019).…”

Section: Introductionmentioning

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: Introductionmentioning

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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“…Further studies on crystal structure of chitinase-substrate complexes may help to elucidate the unique hydrolytic modes of P1724 and its nGH18 and cGH18 domains. Chitinase that have two or three different catalytic modes among endochitinase, exochitinase and N-acetylglucosaminidase is considered to have broad specificity [35]. Compared with the chitinases with single catalytic mode, chitinases with broad specificity have more advantages in lowering the cost in the extraction process and increasing the catalytic efficiencies of the enzymes [35].…”

Section: Discussionmentioning

confidence: 99%

“…Chitinase that have two or three different catalytic modes among endochitinase, exochitinase and N-acetylglucosaminidase is considered to have broad specificity [35]. Compared with the chitinases with single catalytic mode, chitinases with broad specificity have more advantages in lowering the cost in the extraction process and increasing the catalytic efficiencies of the enzymes [35]. Therefore, we believe P1724, P1724nGH18 and P1724cGH18 could have advantages in applications due to their broad specificity.…”

Section: Discussionmentioning

confidence: 99%

Cloning, Purification, and Characterization of a Novel Metagenome-Sourced Microbial Chitinase with Dual Catalytic Domains

Dai

Yang

Liu

et al. 2020

Preprint

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Background: Microbial chitinases have attracted a lot of attention because of their great potential in many applications. Metagenome-based approach obtains the target genes from the environment directly without culturing the microbes and is becoming a powerful tool to discover the novel chitinases. Results: A metagenomic approach was used to discover the chitinases from a wetland on the Qinghai-Tibetan plateau. Gene P1724 was found and predicted to have two GH18 catalytic domains. Gene sequence containing P1724 , only its N-terminal GH18 domain ( P1724nGH18 ) or only C-terminal GH18 domain ( P1724cGH18 ) were cloned and expressed in Escherichia coli BL21 (DE3). These purified recombinant chitinases showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive since they were still active at 4 °C. The activities of three chitinases were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 3+ . All three chitinases showed both chitotiosidase and endochitinase activities, and produced predominantly N, N΄-Diacetylchitobiose from colloid chitin. Other than these common characteristics, P1724 and P1724nGH18 shared more similarity in temperature and pH stabilities, NaCl tolerance and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat / K m value (catalytic efficiency) of P1724 was significantly higher than the sum values of P1724nGH18 and P1724cGH18’s, which indicated that two GH18 domains of P1724 works synergistically in degrading chitin. Conclusion: Compared to the most of microbial chitinases containing only one catalytic domain, chitinases P1724 with two GH18 catalytic domains was discovered firstly by the metagenomic approach. P1724 is a novel chitinase with unique amino acid sequences and hydrolytic mode, and could be used in cold environments or industries.

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“…So far, P1724 has been found to have at least two hydrolytic modes. Compared with the chitinases with single catalytic mode, chitinases with multiple modes have more advantages in lowering the cost in the extraction process and increasing the catalytic efficiencies of the enzymes [37].…”

Section: Discussionmentioning

confidence: 99%

The heterologous expression and characterization of a novel chitinase with dual catalytic domains discovered from a wetland soil metagenome

Dai

Yang

Liu

et al. 2020

Preprint

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Background: Microbial chitinases have attracted a lot of attention because of their great potential in many applications. Metagenome-based approach obtains the target genes from the environment directly without culturing the microbes and is becoming a powerful tool to discover the novel chitinases.Results: Gene P1724 was found in a chitin-enriched microbial community from a wetland on the Qinghai-Tibetan plateau using the metagenomic approach. The translated protein sequence of P1724 was predicted to have two GH18 catalytic domains and showed very low similarities with any known and hypothetical chitinases. The gene sequence of P1724 , its N-terminal GH18 domain ( P1724nGH18 ), or C-terminal GH18 domain ( P1724cGH18 ) was cloned and expressed in Escherichia coli BL21 (DE3). Using colloid chitin as substrate, these purified recombinant chitinases showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive since they were still active at 4 °C; the activities of three chitinases were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 3+ ; all three chitinases showed both chitotiosidase and endochitinase activities, and could hydrolyze chitosan as well. Other than these common characteristics, P1724 and P1724nGH18 shared more similarity in temperature and pH stabilities, NaCl tolerance and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat / K m value (catalytic efficiency) of P1724 was significantly higher than the sum values of P1724nGH18 and P1724cGH18, which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin.Conclusion: Compared to most microbial chitinases that contain only one catalytic domain, P1724 contains two and was firstly discovered by the metagenomic approach. P1724, its N-terminal and C-terminal catalytic domains were heterologously expressed and characterized. These three recombinant chitinases are phylogenetically distant to any chitinases studied so far, have unique hydrolytic mode, high catalytic efficiency and so new promising candidates for chitinases in applications. In addition, this study broadens the knowledge of unknown chitinases in nature and shows a natural strategy for enzyme engineering by adding other catalytic domain(s).

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The broad-specificity chitinases: their origin, characterization, and potential application

Cited by 21 publications

References 24 publications

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

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

Cloning, Purification, and Characterization of a Novel Metagenome-Sourced Microbial Chitinase with Dual Catalytic Domains

The heterologous expression and characterization of a novel chitinase with dual catalytic domains discovered from a wetland soil metagenome

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