High Diversity of β-Glucosidase-Producing Bacteria and Their Genes Associated with Scleractinian Corals

β-Glucosidase is a microbial cellulose multienzyme that plays an important role in the regulation of the entire cellulose hydrolysis process, which is the rate-limiting step in bacterial carbon cycling in marine environments. Despite its importance in coral reefs, the diversity of β-glucosidase-producing bacteria, their genes, and enzymatic characteristics are poorly understood. In this study, 87 β-glucosidase-producing cultivable bacteria were screened from 6 genera of corals. The isolates were assigned to 21 genera, distributed among three groups: Proteobacteria, Firmicutes, and Actinobacteria. In addition, metagenomics was used to explore the genetic diversity of bacterial β-glucosidase enzymes associated with scleractinian corals, which revealed that these enzymes mainly belong to the glycosidase hydrolase family 3 (GH3). Finally, a novel recombinant β-glucosidase, referred to as Mg9373, encompassing 670 amino acids and a molecular mass of 75.2 kDa, was classified as a member of the GH3 family and successfully expressed and characterized. Mg9373 exhibited excellent tolerance to ethanol, NaCl, and glucose. Collectively, these results suggest that the diversity of β-glucosidase-producing bacteria and genes associated with scleractinian corals is high and novel, indicating great potential for applications in the food industry and agriculture.

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Mutagenesis and mechanistic study of a glycoside hydrolase family 54 α-L-arabinofuranosidase from Trichoderma koningii

Biochemical Journal ◽

10.1042/bj20060717 ◽

2006 ◽

Vol 401 (2) ◽

pp. 551-558 ◽

Cited By ~ 14

Author(s):

Chin-Feng Wan ◽

Wei-Hong Chen ◽

Cheng-Ta Chen ◽

Margaret Dah-Tsyr Chang ◽

Lee-Chiang Lo ◽

...

Keyword(s):

Catalytic Activity ◽

Enzymatic Reaction ◽

Glycoside Hydrolase Family ◽

Wild Type ◽

Trichoderma Koningii ◽

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting ◽

Hydrolase Family ◽

Enzyme Intermediate

A GH (glycoside hydrolase) family 54 α-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii α-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl–enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl–enzyme intermediate was proposed. Based on the kcat values of a series of aryl-α-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, βlg=−0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pKa values (>6) of leaving phenols, with βlg=−1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

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Identifiability study of the proteins degradation model, based on ADM1, using simultaneous batch experiments

Water Science & Technology ◽

10.2166/wst.2006.523 ◽

2006 ◽

Vol 54 (4) ◽

pp. 31-39 ◽

Cited By ~ 17

Author(s):

X. Flotats ◽

J. Palatsi ◽

B.K. Ahring ◽

I. Angelidaki

Keyword(s):

Amino Acids ◽

Anaerobic Degradation ◽

Volatile Fatty Acids ◽

Batch Experiments ◽

Degradation Model ◽

Rate Limiting Step ◽

Limiting Step ◽

Hydrolysis Process ◽

Parameter Values ◽

Rate Limiting

The objective of the present study is to analyse kinetic and stoichiometric parameter values of gelatine anaerobic degradation at thermophilic range, based on an experiment designed to elucidate if volatile fatty acids (VFA) are inhibitors of the hydrolysis process. Results showed that VFA are not inhibiting the hydrolysis process. The ADM1 model adequately expressed the consecutive steps of hydrolysis and acidogenesis, with estimated kinetic values corresponding to a fast acidogenesis and slower hydrolysis. The hydrolysis was found to be the rate limiting step of anaerobic degradation. Estimation of yield coefficients based on the relative initial slopes of VFA profiles obtained in a simple batch experiment produced satisfactory results. From the identification study, it was concluded that it is possible to determine univocally the related kinetic parameter values for protein degradation if the evolution of amino acids is measured in simultaneous batch experiments, with different initial protein and amino acids concentrations.

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Effect of some physical and chemical parameters on the fermentation of cellulose to methane by a coculture system

Canadian Journal of Microbiology ◽

10.1139/m83-227 ◽

1983 ◽

Vol 29 (11) ◽

pp. 1475-1480 ◽

Cited By ~ 6

Author(s):

Victoria M. Laube ◽

Stanley M. Martin

Keyword(s):

Cellulose Degradation ◽

Cellulose Hydrolysis ◽

Methanosarcina Barkeri ◽

Chemical Parameters ◽

Fermentation Products ◽

Rate Limiting Step ◽

Limiting Step ◽

Acetate Utilization ◽

Physical And Chemical ◽

Rate Limiting

In the fermentation of cellulose to methane by the triculture of Acetivibrio cellulolyticus – Desulfovibrio sp. – Methanosarcina barkeri, methanogenesis was the rate-limiting step. The optimal temperature was 35 °C. In the presence of initially added hydrogen, initiation of cellulose hydrolysis was delayed until most of the hydrogen was metabolized, and then the fermentation which followed was comparable with the N2: CO2 control. Increased CH4 yields and rates of formation were stoichiometrically related to the utilization of the hydrogen initially present. Added acetate had no effect on cellulolysis. The increased yields of CH4 observed could be accounted for by utilization of the added acetate. When Methanobrevibacter sp. was included in the coculture (without added H2 or acetate), no hydrogen accumulated and lower rates of acetate utilization and subsequent CH4 evolution were observed. These results suggest a requirement for hydrogen by M. barkeri for efficient acetate utilization. Controlling the pH at 6.8 increased the amount of cellulose degraded, but CH4 yields were lower and no acetate was used. Increasing sulfate levels altered the ratio of fermentation products but had no effect on cellulose degradation. Lower CH4 yields were obtained at elevated sulfate concentrations.

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Solid products and rate-limiting step in the thermal half decomposition of natural dolomite in a CO2(g) atmosphere

Thermochimica Acta ◽

10.1016/s0040-6031(02)00603-2 ◽

2003 ◽

Cited By ~ 1

Author(s):

D Beruto

Keyword(s):

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting

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Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646709 ◽

1978 ◽

Vol 39 (02) ◽

pp. 496-503 ◽

Cited By ~ 8

Author(s):

P A D’Amore ◽

H B Hechtman ◽

D Shepro

Keyword(s):

Endothelial Cells ◽

Ornithine Decarboxylase ◽

Decarboxylase Activity ◽

Aortic Endothelial Cells ◽

Ornithine Decarboxylase Activity ◽

Rate Limiting Step ◽

Bovine Aortic Endothelial Cells ◽

Limiting Step ◽

Rate Limiting ◽

Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

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