Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization

Chen, Ping; Fu, Xianyu; Ng, Tzi Bun; Ye, Xiuyun

doi:10.1007/s10529-011-0724-3

Cited by 34 publications

(18 citation statements)

References 15 publications

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“…Number of β-glucosidase genes from bacteria, yeast and fungi have been cloned and expressed in E. coli and eukaryotic systems such as Saccharomyces cerevisiae, Pichia pastoris, and T. reesei [168,180,181,182]. Cloning and expression has started with either 1) genomic DNA digestion, construction of genomic DNA library and then functional screening for β-glucosidase [183], or 2) construction of cDNA library and function-based screening of β-glucosidase [169,184].…”

Section: Cloning and Expression Of Microbial β-Glucosidasesmentioning

confidence: 99%

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Ahmed¹,

Aslam²,

Ashraf³

et al. 2017

JAEM

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Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass.Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases.

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Section: Cloning and Expression Of Microbial β-Glucosidasesmentioning

confidence: 99%

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Ahmed¹,

Aslam²,

Ashraf³

et al. 2017

JAEM

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“…Gupta with coauthors [11] found efficient extracellular secretion of a β glucosidase fused with the hyperosmotically induc ible periplasmic protein, OsmY, in E. coli and exhibited 2.2 U/mL of β glucosidase activity in the extracellular fraction at 16 h post induction. Chen et al [12] reported expression of a secretory β glucosidase from Trichoderma reesei in P. pastoris that the maximum recombinant β glucosidase activity achieved 60 U/mL. Mendoza Aguayo with coworkers [13] revealed extra cellular expression of β glucosidase of Cellulomonas fla vigena PN 120 in the supernatant of a rich medium showing 582 U/L.…”

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confidence: 99%

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Xue

Hou

et al. 2015

Appl Biochem Microbiol

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The β glucosidase Tm bglA gene from hyperthermophile Thermotoga maritima was cloned into expression vectors pET 28a, producing two proteins of 55 kDa and 52 kDa in cell, which could be referred to Tm BglA with and without 23 amino acids. The results showed that fusion of 23 amino acids to Tm BglA improved its soluble expression and product tolerance, resulting over 60% yield of recombinant Tm BglA secreted into the growth medium in Escherichia coli JM109 (DE3). Tm BglA with 23 amino acids had higher k cat and K M values for p nitrophenyl β D glycopyranoside and much stronger product inhibition than Tm BglA without 23 amino acids. Subsequently, the Tm BglA was immobilized on chitin efficiently by genetically fusing the chitin binding domain of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571. The immobilized Tm BglA had higher optimal temperature (95°C) and was more thermostable at the range from 75 to 100°C than its free form. This immobilized protein exhibited high stability and the con version yield exceeding 90%. The high level soluble expression, combined simultaneous purification and immobilization of the enzyme on chitin offers a novel approach for the low cost production of β glucosidase to produce lactose free fresh dairy products.

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“…Based on the diversity in substrate specificity, β-glucosidases have been classified into aryl β-glucosidases that only hydrolyze p-nitrophenyl-β-D-glucopyranoside (pNPG), cellobiase or alkyl β-glucosidases that specifically hydrolyze cello-oligosaccharides (including cellobiose), and broad substrate spectrum β-glucosidases having activity towards both substrates mentioned above [3]. To date, a number of GH3 β-glucosidase genes have been isolated and characterized from different fungi, such as Aspergillus fumigatus [9], Penicillium piceum [10], and Trichoderma reesei [11]. Recently, β-glucosidases from thermophilic fungi have attracted much attention due to its potential applications associated with their inherent biochemical properties like good stability [12].…”

mentioning

confidence: 99%

Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris

Zhao

Guo

Tian

et al. 2015

Appl Biochem Biotechnol

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A novel β-glucosidase of glycoside hydrolase (GH) family 3 from Myceliophthora thermophila (mtbgl3b) was successfully expressed in Pichia pastoris. The full-length gene consists of 2613 bp nucleotides encoding a protein of 870 amino acids. MtBgl3b showed maximum activity at pH 5.0 and remained more than 70 % relative activity at 3.5-6.0. The enzyme displayed the highest activity at 60 °C and kept about 90 % relative activity for 50-65 °C; besides, the enzyme showed psychrophilic trait and remains 51 % relative activity at 40 °C. MtBgl3b exhibited good stability over a wide pH range of 3.0-10.0 and was thermostable at 60 and 65 °C. The enzyme displayed highest activity towards p-nitrophenyl-β-D-glucopyranoside (pNPG), followed by p-nitrophenyl-D-cellobioside (pNPC), cellotetraose, cellotriose, cellobiose, and gentiobiose. When using 10 % cellobiose (w/v) as the substrate, the enzyme showed transglycosylation activity to produce the cellotriose. The kinetic parametric of K m and V max were 2.78 mM and 927.9 μM mg(-1) min(-1), respectively. Finally, the reaction mode of the enzyme and the substrates were analyzed by molecular docking approach.

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Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization

Cited by 34 publications

References 15 publications

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris

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