Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β-Glucosidase BGLII (Cel1A)

ABSTRACT This paper describes the characterization of an intracellular β-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific β-glucosidase, having no β-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this β-glucosidase.

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Phosphagen kinase in Schistosoma japonicum: Characterization of its enzymatic properties and determination of its gene structure

Molecular and Biochemical Parasitology ◽

10.1016/j.molbiopara.2013.04.001 ◽

2013 ◽

Vol 188 (2) ◽

pp. 91-98 ◽

Cited By ~ 10

Author(s):

Shinji Tokuhiro ◽

Kouji Uda ◽

Hiroko Yano ◽

Mitsuru Nagataki ◽

Blanca R. Jarilla ◽

...

Keyword(s):

Schistosoma Japonicum ◽

Gene Structure ◽

Enzymatic Properties ◽

Phosphagen Kinase ◽

Its Gene

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Characterization of chitinases excreted by Bacillus cereus CH

Canadian Journal of Microbiology ◽

10.1139/w99-148 ◽

2000 ◽

Vol 46 (4) ◽

pp. 370-375 ◽

Cited By ~ 7

Author(s):

Naoto Mabuchi ◽

Ichiro Hashizume ◽

Yoshio Araki

Keyword(s):

Bacillus Cereus ◽

Culture Supernatant ◽

Colloidal Chitin ◽

Reaction Products ◽

Amino Terminal ◽

Transglycosylation Activity ◽

Chitinase A ◽

Molecular Masses ◽

Group B

Bacillus cereus CH was shown to excrete chitinases into the culture supernatant when cultivated in a medium containing 0.2% colloidal chitin, whereas the removal of colloidal chitin resulted in a low activity. After concentration of the culture supernatant by precipitation with ammonium sulfate, the induced chitinases were purified by sequential chromatography. Four different chitinases, A, B1, B2, and B3 with molecular masses of 35, 47, 58, and 64 kDa, respectively, were separated. All chitinases showed similarities in their kinetic parameters when observed with colloidal chitin, including an optimal pH of 5.0-7.5, and an optimal temperature between 50-60°C. Chitinase A hydrolyzed glycol chitin and p-nitrophenyl-di-N-acetyl-beta-chitobioside at similar rates to that of colloidal chitin, whereas group B chitinases hydrolyzed both substrates in much lower rates. From analyses of the reaction products, it is most likely that chitinase A and all group B chitinases hydrolyze the substrates tested in an endo-fashion. However, group B chitinases were distinct from chitinase A in possessing high transglycosylation activity. From amino terminal sequencing, chitinases B1, B2, and B3 were shown to have almost identical sequences, which differed from that of chitinase A. The similarities in the reaction modes and amino terminal sequences among chitinases B1, B2, and B3 suggest that these chitinases may be derived from a presumptive precursor protein through C-terminal processing.Key words: Bacillus cereus, chitinase, purification, characterization.

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The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073313 ◽

1973 ◽

Vol 31 ◽

pp. 574-575

Author(s):

J. T. Stasny ◽

R. C. Burns ◽

R. W. F. Hardy

Keyword(s):

Cellular Localization ◽

Direct Evidence ◽

Azotobacter Vinelandii ◽

Intracellular Localization ◽

Protein Component ◽

Electron Microscopic ◽

Thin Sections ◽

Fe Protein ◽

Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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Characterization of the novel aspartic proteinases, BACE and BACE2

Neuroscience Research ◽

10.1016/s0168-0102(00)81592-8 ◽

2000 ◽

Vol 38 ◽

pp. S124

Author(s):

S Kametaka

Keyword(s):

The Novel ◽

Aspartic Proteinases

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Identification and characterization of a novel gene, TrCCD1, and its possible function in hyphal growth and conidiospore development of Trichoderma reesei

Fungal Genetics and Biology ◽

10.1016/j.fgb.2008.12.006 ◽

2009 ◽

Vol 46 (3) ◽

pp. 255-263 ◽

Cited By ~ 6

Author(s):

Yao Hua Zhong ◽

Tian Hong Wang ◽

Xiao Li Wang ◽

Guang Tao Zhang ◽

Hai Na Yu

Keyword(s):

Trichoderma Reesei ◽

Hyphal Growth ◽

Novel Gene ◽

Identification And Characterization

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Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

Biologia ◽

10.2478/s11756-009-0197-1 ◽

2009 ◽

Vol 64 (6) ◽

Cited By ~ 9

Author(s):

Yue-Hong Wang ◽

Yu Jiang ◽

Zuo-Ying Duan ◽

Wei-Lan Shao ◽

Hua-Zhong Li

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Optimal Condition ◽

Industrial Application ◽

Conversion Rate ◽

Hydrolytic Activity ◽

Thermoanaerobacter Ethanolicus ◽

Transglycosylation Activity

AbstractIn this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K m and V max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).

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