Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion

Deng, Jun‐Jin; Shi, Dan; Mao, He-Hua; Li, Zhiwei; Liang, Shuang; Yao, Ke; Luo, Xiao‐Chun

doi:10.1016/j.ijbiomac.2019.04.177

Cited by 72 publications

(60 citation statements)

References 39 publications

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“…This result is in line with observations of chitinase from P. pasadenensis NCIM 5434, where enzymatic activity was completely inhibited by Cu 2+ (Loni et al, 2014). It has been suggested that Cu 2+ catalyzes the autooxidation of cysteine, resulting in the formation of intramolecular disulfide bonds or sulfonic acid, which may inhibit the activity of chitinase (Deng et al, 2019). The influence of EDTA on purified CHI is consistent with chitinase from Chitinolyticbacter meiyuanensis SYBC-H1 (Zhang et al, 2018), whose activity was found to be enhanced by 10 mM EDTA.…”

Section: Discussionsupporting

confidence: 91%

“…At present, degradation products of chitin are usually generated through chemical degradation, which not only has high energy consumption but also creates serious environmental pollution. However, 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). In nature, more than 10 9 tons of chitin is produced each year (Menghiu et al, 2019), and over 10,000 tons of shellfish waste is produced annually, most of which is thrown into the sea or land fill.…”

Section: Introductionmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 91%

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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“…The NACOS generally have not been as commercialized as COS, owing to the high cost associated with the process of re-acetylation from COS by chemical or enzymatic methods [40]. As more efficient chitinases continue to be developed, the traditional methods of NACOS production through chitin deacetylation, the hydrolysis of chitosan, and the re-acetylation of COS would be abandoned and replaced by the direct chitinase hydrolysis of chitin or crustacean shells, which will would facilitate the imminent commercialization of NACOS [32,33]. Until now, the bioactivities of NACOS and their underlying mechanisms have been poorly understood and lagged far behind our understanding of COS mechanisms.…”

Section: Discussionmentioning

confidence: 99%

“…The N-acetyl chitooligosaccharides standards (NACOS2 to NACOS6) were obtained from Megazyme (Bray, Ireland). The NACOS with a low DP (LNACOS) were prepared from colloidal chitin by the chitinase, Chit46, as previously reported [32]. The NACOS with a high DP (HNACOS) were prepared from shrimp shells [33].…”

Section: Materials and Reagentsmentioning

confidence: 99%

Immunomodulatory Effects of N-Acetyl Chitooligosaccharides on RAW264.7 Macrophages

Deng

et al. 2020

Marine Drugs

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The ongoing development of new production methods may lead to the commercialization of N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1–6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1β, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.

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“…At present, degradation products of chitin are usually generated through chemical degradation, which not only has high energy consumption but also creates serious environmental pollution. However, 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). In nature, more than 10 9 tons of chitin is produced each year (Menghiu et al, 2019), and over 10,000 tons of shellfish waste is produced annually, most of which is thrown into the sea or land fill.…”

Section: Introductionmentioning

confidence: 99%

Peer Review #1 of "Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002 (v0.1)"

2020

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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 72.2%. 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 Ca 2+ , Mn 2+ , Tween 80, and urea. Conversely, Cu 2+ , Fe 3+ , 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

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Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion

Cited by 72 publications

References 39 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

Immunomodulatory Effects of N-Acetyl Chitooligosaccharides on RAW264.7 Macrophages

Peer Review #1 of "Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002 (v0.1)"

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