Purification, Biochemical Characterization of a Macrotermes gilvus Cellulase and Zymogram Analysis

Champasri, Chamaiporn; Champasri, Thongchai; Woranam, Khanutsanan

doi:10.3923/ajb.2015.190.204

Cited by 12 publications

(2 citation statements)

References 48 publications

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“…Another study recorded that metal ions such as Na + (sodium ion), K + (potassium ion), Ca 2+ (Calcium ion), Mg 2+ (Magnesium ion) and Mn 2+ (Manganese ion) improved the cellulase activity produced by B. amyoliquefaciens DL-3. Besides, the presence of Ca 2+ for Clostridium thermocellum , D-endoglucanase is reported to stabilise the enzyme against thermal denaturation and increase substrate binding affinity (Champasri et al . 2015).…”

Section: Resultsmentioning

confidence: 99%

Isolation and Partial Characterisation of Thermophilic Cellulolytic Bacteria from North Malaysian Tropical Mangrove Soil

Naresh

Kunasundari

Gunny

et al. 2019

tlsr

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This study reports the biodiversity of thermophilic cellulolytic bacterial strains that present in the north Malaysian mangrove ecosystem. Soil samples were collected at the four most northern state of Malaysia (Perak, Pulau Pinang, Kedah and Perlis). The samples obtained were first enriched in nutrient broth at 45°C and 55°C prior culturing in the carboxymethylcellulose (CMC) agar medium. Repeated streaking was performed on the CMC agar to obtain a pure culture of each isolate prior subjecting it to hydrolysis capacity testing. The isolates that showing the cellulolytic zone (halozone) were sent for 16S rRNA sequencing. Total seven isolates (two from Perak, three from Kedah, another two were from Perlis and Penang each) showed halozone. The isolate (KFX-40) from Kedah exhibited highest halozone of 3.42 ± 0.58, meanwhile, the one obtained from Perak (AFZ-0) showed the lowest hydrolysis capacity (2.61 ± 0.10). Based on 16S rRNA sequencing results, 5 isolates (AFY-40, AFZ-0, KFX-40, RFY-20, and PFX-40) were determined to be Anoxybacillus sp . The other two isolates were identified as Bacillus subtilis (KFY-40) and Paenibacillus dendritiformis (KFX-0). Based on growth curve, doubling time of Anoxybacillus sp . UniMAP-KB06 was calculated to be 32.3 min. Optimal cellulose hydrolysis temperature and pH of this strain were determined to be 55°C and 6.0 respectively. Addition of Mg 2+ and Ca 2+ were found to enhance the cellulase activity while Fe 3+ acted as an enzyme inhibitor.

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Section: Resultsmentioning

confidence: 99%

Isolation and Partial Characterisation of Thermophilic Cellulolytic Bacteria from North Malaysian Tropical Mangrove Soil

Naresh

Kunasundari

Gunny

et al. 2019

tlsr

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“…In-gel assays for cellulase activity were carried out as detailed by Cano-Ramı ´rez et al [24] and Champasri et al [25] with slight modifications: HrGH45 was loaded onto a 4% stacking gel without any reducing agents or heating. Moreover, CMC was incorporated into the 12% resolving phase gel at a final concentration of 0.1% (w/v).…”

Section: Zymographymentioning

confidence: 99%

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity

Hernández-Benítez,

Ramírez-Rodríguez,

Hernández-Santoyo

et al. 2024

PLoS ONE

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The red abalone (Haliotis rufescens) represents North America’s most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-β-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a β-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

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Purification and Characterization of the Total Cellulase Activities (TCA) of Cellulolytic Microorganisms

Kameshwar

Qin

2018

Cellulases

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Cellulose is the earth's most abundant plant polysaccharide containing a large array of glucose units linked through β (1 → 4) linkages by existing in both crystalline and amorphous forms. Cellulose is widely distributed in plants, constituting up to 40-50% overall dry weight of the plant biomass. Majorly, microorganisms secrete three types of enzymes such as endoglucanases, exoglucanases, and beta-glucosidase for the hydrolysis of cellulose, contributing to the total cellulase activity. Industrially, the cellulolytic microorganisms are assessed based on their total cellulolytic activities. Similarly, total cellulase activity can also be used for the isolation and characterization of the cellulolytic microorganisms. In this chapter, we have specifically discussed about the methods used for the purification and characterization of the total cellulase activities of the microorganisms such as filter paper assay and cellulase zymogram assay. Our present chapter can be used as primer for characterizing cellulolytic abilities of cellulose-degrading microorganisms.

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Purification, Biochemical Characterization of a Macrotermes gilvus Cellulase and Zymogram Analysis

Cited by 12 publications

References 48 publications

Isolation and Partial Characterisation of Thermophilic Cellulolytic Bacteria from North Malaysian Tropical Mangrove Soil

Isolation and Partial Characterisation of Thermophilic Cellulolytic Bacteria from North Malaysian Tropical Mangrove Soil

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity

Purification and Characterization of the Total Cellulase Activities (TCA) of Cellulolytic Microorganisms

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