Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

Chen, Chun-Ti; Huang, Chang‐Jen; Wang, Yi-Huei; Chen, Chao‐Ying

doi:10.1016/j.pep.2004.03.017

Cited by 6 publications

(3 citation statements)

References 26 publications

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“…This result showed the maximum chitinase was precipitated at 40% salt concentration. This result was in co-relation with the result obtained by Chen et al (2004) during the two step purification of the Bacillus circulans chitinase A expressed in E. coli periplasm. Similarly, the putative chitinase band (88kDa) with 40% ammonium sulphate fractionation gave the highest yield of the desired protein.…”

Section: Inhibition Of the Phytopathogenssupporting

confidence: 84%

Invitro Screening of Antifungal Activity of Rhizospheric Bacteria and Possible Role of Chitinase in Antifungal Activity

Devkota

Maharjan

Baral

et al. 2012

Nepal Journal of Science and Technology

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The use of biocontrol agents is becoming an increasingly important alternative to chemical crop protection against weeds, insects and plant diseases in the field of agriculture. The success of biocontrol and yield increase depends on the nature of antagonistic properties and mechanisms of action of the biocontrol agent against the phytopathogens. In this study, 103 macroscopically different bacterial isolates (62 from Kirtipur and 41 from Khumaltar) from 21 different rhizosphere soil samples (11 from Kirtipur and 10 from Khumaltar) were screened for antagonism against five fungal phytopathogens, viz, Fusarium oxysporum, Alternaria solani, Sclerotium rolfsii, Exserohilum turcicum and Phytophthora infestans by dual culture technique on Potato dextrose agar. Out of 18 different active isolates two of them showed the chitinolytic potential and the most active fungal antagonist was identified as Bacillus subtilis on the basis of colonial, morphological, physiological and biochemical characteristics based on Bergey’s Manual of systemic bacteriology. The isolate produced maximum chitinase in colloidal chitin broth at pH7 and temperature 37oC after four days of inoculation. The corresponding culture filtrate supposed to contain chitinase showed maximum % inhibition of 53.29% with Fusarium oxysporum and no inhibition to Phytophthora infestans in agar well diffusion assay. Furthermore, chitinase was best fractionated at 40% ammonium sulphate salt fractionation which has almost similar inhibition potential as the crude culture filtrate. The 40% salt fraction of the enzyme showed the maximum chitinolytic potential at pH8 and temperature 40oC. Among the phytopathogens tested, sensitivity of Bacillus subtilis to fungi containing chitin on their cell wall demonstrates the possible role of chitinase in the antifungal activity.DOI: http://dx.doi.org/10.3126/njst.v12i0.6517 Nepal Journal of Science and Technology 12 (2011) 304-311

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Section: Inhibition Of the Phytopathogenssupporting

confidence: 84%

Invitro Screening of Antifungal Activity of Rhizospheric Bacteria and Possible Role of Chitinase in Antifungal Activity

Devkota

Maharjan

Baral

et al. 2012

Nepal Journal of Science and Technology

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“…8.3 mg/L for ChiA01 [ 26 ]. Notably, Chi18H8 purification yield also favorably compared with those obtained for other recombinant chitinolytic enzymes produced in E. coli : only few micrograms per liter of culture were obtained in the case of Bacillus circulans WL-12 ChiA (95 μg/L) [ 49 ], whereas milligrams of protein were prepared for HschiA1 from Halobacterium salinarum CECT 395 (1.4 mg/L) [ 50 ], for ChiCH and ChiCW from Bacillus cereus (4.6 and 2.3 mg/L, respectively) [ 51 ], for Bacillus licheniformis ChiA (20 mg/L) [ 52 ] and for Paecilomyces thermophila PtChiA (28 mg/L) [ 53 ].…”

Section: Discussionmentioning

confidence: 99%

Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome

Berini

Presti

Beltrametti³

et al. 2017

Microb Cell Fact

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BackgroundThrough functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase—named Chi18H8 and belonging to family 18 glycosyl hydrolases—was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 μg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential.ResultsWe report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate.ConclusionsExpression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0634-8) contains supplementary material, which is available to authorized users.

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“…Serratia marcescens produces three chitinases and one chitin-binding protein (CBP) (41). Bacillus circulans secretes three chitinases (5) in the presence of chitin. Most of the bacterial chitinases have a multidomain organization, but a few have only a catalytic domain, for example, ChiCH from B. cereus 28-9 (16) and ChiB from Stenotrophomonas maltophilia (K. Suma and A. R. Podile, unpublished data).…”

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confidence: 99%

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

Purushotham

Podile

2012

J Bacteriol

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We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40°C, where it showed a K m of 83 mg ml ؊1 , a k cat of 3.9 ؋ 10 2 h ؊1 , and a k cat /K m of 4.7 h mg ؊1 ml ؊1 on colloidal chitin. On chitobiose, the K m , k cat , and k cat /K m were 203 M, 1.3 ؋ 10 2 h ؊1 , and 0.62 h ؊1 M ؊1 , respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.

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Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

Cited by 6 publications

References 26 publications

Invitro Screening of Antifungal Activity of Rhizospheric Bacteria and Possible Role of Chitinase in Antifungal Activity

Invitro Screening of Antifungal Activity of Rhizospheric Bacteria and Possible Role of Chitinase in Antifungal Activity

Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

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