Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

Priyadharshini Ramachandran; Manish Kumar  Tiwari; Raushan Kumar Singh; Jung-Rim Haw; Marimuthu Jeya; Jung-Kul Lee

doi:10.1016/j.procbio.2011.10.015

Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

Priyadharshini Ramachandran, Manish Kumar Tiwari, Raushan Kumar Singh, Jung-Rim Haw, Marimuthu Jeya, Jung-Kul Lee

Konkuk University

Research output: Contribution to journal › Journal article › Research › peer-review

Abstract

Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with Vmax of 1693Umg-protein−1 and a Km of 2.8mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40°C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.

Original language	English
Journal	Process Biochemistry
Volume	47
Issue number	1
Pages (from-to)	99-105
Number of pages	7
ISSN	1359-5113
DOIs	https://doi.org/10.1016/j.procbio.2011.10.015
Publication status	Published - 2012
Externally published	Yes

Keywords

Characterization
β-Glucosidase
Glycoside hydrolase family 1
Molecular dynamics simulation
Wine aroma

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title = "Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri",

abstract = "Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with Vmax of 1693Umg-protein−1 and a Km of 2.8mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40°C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.",

keywords = "Characterization, β-Glucosidase, Glycoside hydrolase family 1, Molecular dynamics simulation, Wine aroma",

author = "Priyadharshini Ramachandran and Tiwari, {Manish Kumar} and Singh, {Raushan Kumar} and Jung-Rim Haw and Marimuthu Jeya and Jung-Kul Lee",

year = "2012",

doi = "10.1016/j.procbio.2011.10.015",

language = "English",

volume = "47",

pages = "99--105",

journal = "Process Biochemistry",

issn = "1359-5113",

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number = "1",

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TY - JOUR

T1 - Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

AU - Ramachandran, Priyadharshini

AU - Tiwari, Manish Kumar

AU - Singh, Raushan Kumar

AU - Haw, Jung-Rim

AU - Jeya, Marimuthu

AU - Lee, Jung-Kul

PY - 2012

Y1 - 2012

N2 - Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with Vmax of 1693Umg-protein−1 and a Km of 2.8mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40°C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.

AB - Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with Vmax of 1693Umg-protein−1 and a Km of 2.8mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40°C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.

KW - Characterization

KW - β-Glucosidase

KW - Glycoside hydrolase family 1

KW - Molecular dynamics simulation

KW - Wine aroma

U2 - 10.1016/j.procbio.2011.10.015

DO - 10.1016/j.procbio.2011.10.015

M3 - Journal article

SN - 1359-5113

VL - 47

SP - 99

EP - 105

JO - Process Biochemistry

JF - Process Biochemistry

IS - 1

ER -

Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

Abstract

Keywords

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