Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana

Tak Hyun Park, Ki Won Choi, Cheon Seok Park, Soo Bok Lee, Ho Young Kang, Kwang Jae Shon, Jang Su Park, Jaeho Cha

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.

Original languageEnglish
Pages (from-to)411-422
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume69
Issue number4
DOIs
Publication statusPublished - 2005 Dec 1

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Thermotoga neapolitana
Glucosidases
Cellobiose
Substrate Specificity
Arbutin
Enzymes
Glucosides
Mass Spectrometry
Eubacterium
Glucose
Mercaptoethanol
Dithiothreitol
Recycling
Lactose
Thin Layer Chromatography
Affinity Chromatography
Half-Life
Magnetic Resonance Spectroscopy
Hot Temperature
Metals

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Park, Tak Hyun ; Choi, Ki Won ; Park, Cheon Seok ; Lee, Soo Bok ; Kang, Ho Young ; Shon, Kwang Jae ; Park, Jang Su ; Cha, Jaeho. / Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana. In: Applied Microbiology and Biotechnology. 2005 ; Vol. 69, No. 4. pp. 411-422.
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abstract = "The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94{\%} of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.",
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Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana. / Park, Tak Hyun; Choi, Ki Won; Park, Cheon Seok; Lee, Soo Bok; Kang, Ho Young; Shon, Kwang Jae; Park, Jang Su; Cha, Jaeho.

In: Applied Microbiology and Biotechnology, Vol. 69, No. 4, 01.12.2005, p. 411-422.

Research output: Contribution to journalArticle

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T1 - Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana

AU - Park, Tak Hyun

AU - Choi, Ki Won

AU - Park, Cheon Seok

AU - Lee, Soo Bok

AU - Kang, Ho Young

AU - Shon, Kwang Jae

AU - Park, Jang Su

AU - Cha, Jaeho

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Y1 - 2005/12/1

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