Overexpression of β-glucosidase from Thermotoga maritima for the production of highly purified aglycone isoflavones from soy flour

Xue, Yemin; Song, Xiangfei; Yu, Jinjin

doi:10.1007/s11274-009-0121-4

Cited by 20 publications

(26 citation statements)

References 25 publications

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“…These results showed that the intrinsic bioactivity of the Tm BglA2 likely to be improved by fusing 23 amino acids, which is con firmed by the circular dichroism spectra that the sec ondary structure of Tm BglA2 was greatly altered by 23 amino acids fusion (data not shown). These data are comparable to those reported previously [4,5] that the addition of a six histidine tag at the amino termi nal of Tm BglA produced a less catalytic efficiency.…”

Section: Discussionsupporting

confidence: 91%

“…Thermatoga maritima grows optimally at 80°C and has 2 hyperthermostable β glucosidases (Tm BglA and Tm BglB) [1,2] currently used in many industrial applications [3][4][5]. The Tm BglA belongs to glycoside hydrolase family 1 β glucosidases (GH1) and cataly ses the hydrolysis of β glycosidic bonds with wide sub strate specificity.…”

mentioning

confidence: 99%

“…Like the GH1 family enzymes, it contains the highly conserved peptide motifs TLNEP and ITENG including the two catalytic residues of Glu, E166 and E351, located in β strands, 4 and 7, which make up part of a crater shaped active site. In a previous study, the Tm bglA gene from T. maritma was over expression in Escherichia coli, resulted in the pro duction of large amount of inclusion bodies with a weak β glucosidase activity [4]. Therefore, in view of the commercial importance of β glucosidase enzymes, effective enzyme conversion of lactose with high pro ductivity and low cost should attract considerable atten tion, it is thus necessary to produce soluble Tm BglA in large amounts.…”

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confidence: 99%

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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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Section: Discussionsupporting

confidence: 91%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…For substrate specificity, TthBgl only hydrolyzed p-nitrophenyl-β-D-glucopyranoside, while β-glucosidases from T. maritima MSB8 and T. petrophila RKU-1 (ABQ46916) hydrolyze several types of pNP-glycosides (Gabelsberger et al 1993;Cota et al 2015). In terms of Km, TthBgl had a value of 2.41 mM for pNPG, which was lower than that of T. petrophila RKU-1 (ABQ46970) (2.8 mM) but higher than that of T. maritima MSB8 (0.43 mM) and T. petrophila RKU-1 (ABQ46916) (0.38 ± 0.03 mM) (Xue et al 2009;Haq et al 2012;Cota et al 2015).…”

Section: Multiple Sequence Alignment and Phylogenetic Analysis Of Tthbglmentioning

confidence: 90%

“…Data shown are from one typical experiment that was repeated three times, and the variation about the mean was below 5%. β-glucosidase from Thermofilum pendens maintained 95% activity after incubation for 120 min at 90 ºC , and the residual activity of His-tagged β-glucosidase from T. maritima is 75% after being incubated at 80 ºC for 60 min (Xue et al 2009). The highly thermostable β-glucosidase from T. petrophila RKU-1 (ABQ46970) retains 80% activity after incubation for 120 min at 80 ºC (Haq et al 2012).…”

Section: Biochemical and Kinetic Parametersmentioning

confidence: 99%

Cloning, Purification, and Characterization of a Thermostable β-Glucosidase from Thermotoga thermarum DSM 5069

Long¹,

Shi²,

Li³

et al. 2016

BioResources

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A 56-kDa β-glucosidase (TthBgl) derived from Thermotoga thermarum DSM 5069 was expressed and purified from Escherichia coli BL21 (DE3). The purified enzyme showed hydrolytic activity towards only pnitrophenyl-β-D-glucopyranoside among the synthetic glycosides tested. The pH maximum was 5.0, and under the conditions tested, maximal activity was at 85 ºC, and pH stability occurred from 5.0 to 6.0. After being incubated at 80 ºC for 120 min, TthBgl retained 80% of its original activity. The β-glucosidase had no apparent requirement for metal ions or other co-factors, but its activity was significantly inhibited by 0.1% SDS and 1mM Cu 2+ , in which only 3% and 10% residual activity was maintained, respectively. The Vmax of TthBgl was 8.79 U mg -1 for pnitrophenyl-β-D-glucopyranoside, while the Km was 2.41 mM. The Enzyme activity was gradually inhibited by the addition of glucose, but remained approximately 50% of its original value in 500 mM glucose. 789.25 mg/L glucose was released from cellobiose by the incubation of 0.2 U/mL TthBgl for 9 h at 75 ºC. According to a phylogenetic analysis, TthBgl belongs to the glycosyl hydrolase family 3 (GH3).

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Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state

Wang

Song

Zhang

et al. 2015

Biotech & Bioengineering

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Trichoderma reesei (Tr.) cellulases, which convert cellulose to reducing sugars, are a promising catalyst used in the lignocellulosic biofuel production. Improving Tr. cellulases activity, though very difficult, is highly desired due to the recalcitrance of lignocellulose. Meanwhile, it is preferable to enhance the cellulase's promiscuity so that substrates other than cellulose can also be hydrolyzed. In this work, an attempt is made to improve the catalytic activity of a major endogluanase Tr. Cel7B against xylan which crosslinks with cellulose in lignocellulose. By using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the transition state of the xylo-oligosaccharide hydrolysis is identified. Then, mutations are introduced and their effect on the transition state stabilization is ranked based on the free energy calculations. Seven top ranked mutants are evaluated experimentally. Three mutants A208Q, A222D, and G230R show a higher activity than the wild-type Tr. Cel7B in the hydrolysis of xylan (by up to 47%) as well as filter paper (by up to 50%). The combination of the single mutants can further improve the enzyme activity. Our work demonstrates that the free energy method is effective in engineering the Tr. Cel7B activity against xylan and cellulose, and thus may also be useful for improving the activity of other Tr. cellulases. Biotechnol. Bioeng. 2016;113: 1171-1177. © 2015 Wiley Periodicals, Inc.

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Overexpression of β-glucosidase from Thermotoga maritima for the production of highly purified aglycone isoflavones from soy flour

Cited by 20 publications

References 25 publications

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

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

Cloning, Purification, and Characterization of a Thermostable β-Glucosidase from Thermotoga thermarum DSM 5069

Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state

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