Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides

Katrolia, Priti; Jia, Honglei; Yan, Qiaojuan; Song, Shuang; Jiang, Zhengqiang; Xu, Hui

doi:10.1016/j.biortech.2012.01.144

Cited by 80 publications

(64 citation statements)

References 29 publications

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“…Raffinose was almost completely hydrolyzed to galactose and sucrose in just 30 min, while only a small amount of stachyose was hydrolyzed. The ability to rapidly hydrolyze raffinose could be explained by the fact that the enzyme displayed greater substrate specificity and catalytic efficiency for this sugar as compared to stachyose, which was different from the results from Priti katrolia, showing higher binding affinity to stachyose [12]. There are some reports which showed that alpha-galactosidases from other sources degraded RFOs.…”

Section: Enzymatic Hydrolysis Of Oligosaccharidesmentioning

confidence: 89%

“…The hydrolysates were then analyzed qualitatively by thin-layer chromatography (TLC). The reaction mixtures were spotted onto a silica gel plate (Merck Silica Gel 60 F 254 , Germany) and developed by ascending chromatography using containing propanol/acetic acid/water at 1:1.5:0.1 (v/v/v) [12] as the solvent system. The sugar spots were located by heating in an electric plate after spraying a mixture of diphenylamine/ aniline/acetone/phosphate (1:1:50:10, m/v/v/v).…”

Section: Enzymatic Hydrolysis Of Oligosaccharidesmentioning

confidence: 99%

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A Fungal Alpha-Galactosidase from Pseudobalsamia microspora Capable of Degrading Raffinose Family Oligosaccharides

Yang

Gao

et al. 2015

Appl Biochem Biotechnol

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An alpha-galactosidase was purified from Pseudobalsamia microspora (PMG) to 1224.1-fold with a specific activity of 11,274.5 units/mg by ion-exchange chromatography and gel filtration. PMG is a monomeric protein with a molecular mass of 62 kDa as determined by SDS-PAGE and by gel filtration. Chemical modification using N-bromosuccinimide (NBS) resulted in a complete abrogation of the activity of PMG, suggesting that Trp is an amino acid essential to its activity. The activity was strongly inhibited by Hg(2+), Cd(2+), Cu(2+), and Fe(3+) ions. Three inner peptide sequences for PMG were obtained by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. When 4-nitrophenyl α-D-glucopyranoside (pNPGal) was used as substrate, the optimum pH and temperature of PMG were 5.0 and 55 °C, respectively. The Michaelis constant (K m) value of the alpha-galactosidase on pNPGal was 0.29 mM, and the maximal velocity (V max) was 0.97 μmol ml(-1) min(-1). Investigation by thin-layer chromatography (TLC) demonstrated its ability to hydrolyze raffinose and stachyose. Hence, it can be exploited in degradation of non-digestible oligosaccharides from food and feed industries.

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Section: Enzymatic Hydrolysis Of Oligosaccharidesmentioning

confidence: 89%

Section: Enzymatic Hydrolysis Of Oligosaccharidesmentioning

confidence: 99%

A Fungal Alpha-Galactosidase from Pseudobalsamia microspora Capable of Degrading Raffinose Family Oligosaccharides

Yang

Gao

et al. 2015

Appl Biochem Biotechnol

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“…In the present work, a novel gene encoding the L-asparaginase from Rhizomucor miehei CAU432, a strain of thermophilic fungus that thrives at an optimum temperature of 50°C (15), was cloned. The heterologous expression, purification, and characterization of this recombinant L-asparaginase (RmAsnase) are described.…”

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confidence: 99%

Biochemical Characterization of a Novel l -Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

Huang

Liu

Sun

et al. 2014

Appl Environ Microbiol

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A novel fungal gene encoding the Rhizomucor miehei L-asparaginase (RmAsnase) was cloned and expressed in Escherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported L-asparaginases. The purified L-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this L-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

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“…It is common that a-galactosidases exhibit higher activities against synthetic substrates (e.g., pNPGal) than natural substrates (melibiose, raffinose and stachyose) (17,30,31).…”

Section: Enzyme Properties Of Purified Raga27amentioning

confidence: 99%

Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity

Wang

et al. 2016

Journal of Bioscience and Bioengineering

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Thermophilic α-galactosidases have great potentials in biotechnological and medicinal applications due to their high-temperature activity and specific stability. In this study, a novel α-galactosidase gene of glycoside hydrolase family 27 (aga27A) was cloned from Talaromyces leycettanus JCM12802 and successfully expressed in Pichia pastoris GS115. Purified recombinant Aga27A (rAga27A) was thermophilic and thermotolerant, exhibiting the maximum activity at 70°C and retaining stability at 65°C. Like most fungal α-galactosidases, rAga27A had an acidic pH optimum (pH 4.0) but retained stability over a boarder pH range (pH 3.0-11.0) at 70°C. Moreover, the enzyme exhibited strong resistance to most metal ions and chemicals tested (except for Ag(+) and SDS) and great tolerance to galactose (19 mM). The preferable transglycosylation capacity of rAga27A with various substrates further widens its application spectrum. Thus rAga27A with excellent enzymatic properties will be ideal for applications in various industries, especially for the synthesis of galactooligosaccharides.

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Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides

Cited by 80 publications

References 29 publications

A Fungal Alpha-Galactosidase from Pseudobalsamia microspora Capable of Degrading Raffinose Family Oligosaccharides

A Fungal Alpha-Galactosidase from Pseudobalsamia microspora Capable of Degrading Raffinose Family Oligosaccharides

Biochemical Characterization of a Novel l -Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity

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