Characterization of a novel cellobiase fromBacillus subtilis and expression of its structural gene inEscherichia coli

Srivastava, Ranjana; Bharti, Rajnish; Srivastava, Alok Kumar

doi:10.1007/bf01086350

Cited by 4 publications

(2 citation statements)

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“…Low transcription levels caused by the inability of E. coli RNA polymerase to recognize the M. bispora bglB promoter could account for the relatively low specific activity of cloned Bg1B in the crude extracts compared with that in enzyme extracts from M. bispora (0.13 and 5.0, respectively). Nevertheless, BglB expression (in a crude protein extract) was comparable to that of products of other cloned bacterial ,B-glucosidase genes which exhibit specific activities for cellobiose that range from 0.08 to 1.83 (2,8,13,17,23,34,37,43,44). The increase in BglB activity exhibited by subclones carrying the smaller (2.2-kb) genomic fragment might have resulted from deletion of a negative regulator element within the larger fragment (9.5 kb).…”

Section: Gagctcggtagccctggtgtagacgggccgcatcgacggctgcggaagctggcggctgctmentioning

confidence: 83%

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli

Wright

Yablonsky

et al. 1992

Appl Environ Microbiol

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Genomic DNA frgments encoding 1-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing e-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbeliiferyl-o-D-glucoside. Two genes encoding 13-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a 13-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60°C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-f-D-glucosides and was thermostable, retaining about 70%o of its activity after 48 h at 60°C. BglB activity is activated two-to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bgiB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with 13-glucosidases from glycosal hydrolase family 1. Cellulose, an abundant but recalcitrant biopolymer, is composed of repeating glucose units linked by P-1,4-glycosidic bonds. Cellulases, produced by a wide variety of microorganisms, degrade such polymers and play a major role in the recycling of biomass. Cellulases are multicomponent complexes which are often composed of endoglucanases (1,4-0-D-glucan glucano-hydrolase, EC 3.2.1.4), cellobiohydrolases (1,4-0-D-glucan cellobiohydrolase, EC 3.2. 1.91), and cellobiases (1,4-0-D-glucoside glucohydrolases, EC 3.2.1.21). Cellobiases, while specific for cellobiose, belong to a very diverse family of enzymes (,-glucosidases) capable of hydrolyzing a broad spectrum of ,-glucosides. Cellulase components are thought to act in a stepwise process and can act synergistically to achieve more efficient degradation (10, 53, 54, 56). The major end product of concerted endoglucanase and cellobiohydrolase activity is cellobiose. Cellobiose is then hydrolyzed to glucose by * Corresponding author. 34), and certain of the genes have been cloned and characterized in heterologous hosts (15, 28, 37). To obtain microorganisms that produce 3-glucosidases that are both thermostable and resistant to end product inhibition, we isolated thermophilic cellulolytic actinomycetes from thermal ecological niches and evaluated their enzymes by assaying for ,B-glucosidase activity (against para-nitrophenyl-1-glucoside) in the presence of up to 30% glucose at 60°C (49). Of several isolates, the most attractive was Microbispora bispora (31, 49). In order to study the enzyme components independently and to characterize the genes encoding them, we have cloned several M. bispora cellulase components (56). These include t...

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Section: Gagctcggtagccctggtgtagacgggccgcatcgacggctgcggaagctggcggctgctmentioning

confidence: 83%

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli

Wright

Yablonsky

et al. 1992

Appl Environ Microbiol

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“…Gravity drainage tests were performed using de-ionized water and solutions of carboxy methyl cellulose (CMC) to simulate the oil phase with different viscosity values (Srivastava et al 1990;Srivastava et al 1992), and air to simulate the gas phase. Viscosity was increased by adding CMC in various concentrations using a blender to achieve full dissolution and homogeneity.…”

Section: Experimental Workmentioning

confidence: 99%

Experimental Study of Controlled Gravity Drainage in Fractured Porous Media

Zendehboudi

Chatzis

2011

Journal of Canadian Petroleum Technology

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Oil production from a fractured reservoir with a gas cap and an oil zone usually takes place at a constant withdrawal rate until gas breakthrough; thereafter, the pumping rate is influenced by the presence of gas. Knowing the effect of pumping rate on production performance before gas breakthrough and selecting optimum pumping rates are necessary. In this paper, the concept of "critical production rate" (CPR) is introduced; it is the production rate at which a porous medium has a recovery factor (RF) equal to that for higher rates before gas enters into the production well, and is also the rate above which the difference between liquid levels in the fracture and matrix remains unchanged. One may use the CPR to choose a lower pumping rate to increase RF before gas breakthrough and to aid in understanding the physics of pumping from fractured media in real cases.Flow visualization experiments were performed using rectangular unconsolidated-packed models with two fractures on the sides. Sensitivity analyses were performed on the effects of different system parameters on the CPR, the maximum possible withdrawal rate (MPWR), RF at gas breakthrough, and gas-liquid (G-L) interface behaviour in both matrix and fractures. Results show that as long as the porous medium is drained with a constant liquid withdrawal rate below CPR, the height difference between G-L interfaces in the matrix and fracture remains constant. CPR and RF can also be related to other system parameters using dimensionless numbers, such as fracture/matrix permeability ratio and Bond number.

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Characterization of a bacterial xylanase resistant to repression by glucose and xylose

Srivastava

1993

Biotechnol Lett

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Characterization of a novel cellobiase fromBacillus subtilis and expression of its structural gene inEscherichia coli

Cited by 4 publications

References 10 publications

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli

Experimental Study of Controlled Gravity Drainage in Fractured Porous Media

Characterization of a bacterial xylanase resistant to repression by glucose and xylose

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