Capacity of thermomonospora alba Xy1A to impart thermostability in family F/10 chimeric xylanases

Mohammad Mainul Ahsan, Satoshi Kaneko, Qin Wang, Kei Yura, Mitiko Go, Kiyoshi Hayash

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and Xy1A of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-Xy1A, FXYN-M9/10-Xy1A, and FXYN-M14/15-Xy1A were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic Xy1A. Measured K(m) and kcat values were closer to those of the other parent, Xy1A. Interestingly, a significant level of heat tolerance up to 60°C, was recorded for FXYN-M3/4-Xy1A in comparison to only 40°C for FXYN-M14/15-Xy1A though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba Xy1A DNA fragment present in FXYN-M3/4-Xy1A were responsible for inducing its thermal stability. (C) 2001 Elsevier Science Inc.

Original languageEnglish
Pages (from-to)8-15
Number of pages8
JournalEnzyme and Microbial Technology
Volume28
Issue number1
DOIs
Publication statusPublished - 2001 Jan 2
Externally publishedYes

Fingerprint

Amino acids
Hot Temperature
Amino Acids
Thermodynamic stability
Proteins
Xylans
Polymerase chain reaction
Streptomyces
Nucleotides
DNA
Fusion reactions
Genes
Polymerase Chain Reaction
Temperature
Experiments
glycanase
Thermotolerance
In Vitro Techniques

Keywords

  • Chimeric xylanases
  • Family 10 xylanases
  • Module shuffling
  • Streptomyces olivaceoviridis
  • Thermomonospora alba

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Capacity of thermomonospora alba Xy1A to impart thermostability in family F/10 chimeric xylanases. / Ahsan, Mohammad Mainul; Kaneko, Satoshi; Wang, Qin; Yura, Kei; Go, Mitiko; Hayash, Kiyoshi.

In: Enzyme and Microbial Technology, Vol. 28, No. 1, 02.01.2001, p. 8-15.

Research output: Contribution to journalArticle

Ahsan, Mohammad Mainul ; Kaneko, Satoshi ; Wang, Qin ; Yura, Kei ; Go, Mitiko ; Hayash, Kiyoshi. / Capacity of thermomonospora alba Xy1A to impart thermostability in family F/10 chimeric xylanases. In: Enzyme and Microbial Technology. 2001 ; Vol. 28, No. 1. pp. 8-15.
@article{bf9ff86fc6d440818a70e20e53266a7d,
title = "Capacity of thermomonospora alba Xy1A to impart thermostability in family F/10 chimeric xylanases",
abstract = "To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and Xy1A of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-Xy1A, FXYN-M9/10-Xy1A, and FXYN-M14/15-Xy1A were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic Xy1A. Measured K(m) and kcat values were closer to those of the other parent, Xy1A. Interestingly, a significant level of heat tolerance up to 60°C, was recorded for FXYN-M3/4-Xy1A in comparison to only 40°C for FXYN-M14/15-Xy1A though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba Xy1A DNA fragment present in FXYN-M3/4-Xy1A were responsible for inducing its thermal stability. (C) 2001 Elsevier Science Inc.",
keywords = "Chimeric xylanases, Family 10 xylanases, Module shuffling, Streptomyces olivaceoviridis, Thermomonospora alba",
author = "Ahsan, {Mohammad Mainul} and Satoshi Kaneko and Qin Wang and Kei Yura and Mitiko Go and Kiyoshi Hayash",
year = "2001",
month = "1",
day = "2",
doi = "10.1016/S0141-0229(00)00279-9",
language = "English",
volume = "28",
pages = "8--15",
journal = "Enzyme and Microbial Technology",
issn = "0141-0229",
publisher = "Elsevier Inc.",
number = "1",

}

TY - JOUR

T1 - Capacity of thermomonospora alba Xy1A to impart thermostability in family F/10 chimeric xylanases

AU - Ahsan, Mohammad Mainul

AU - Kaneko, Satoshi

AU - Wang, Qin

AU - Yura, Kei

AU - Go, Mitiko

AU - Hayash, Kiyoshi

PY - 2001/1/2

Y1 - 2001/1/2

N2 - To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and Xy1A of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-Xy1A, FXYN-M9/10-Xy1A, and FXYN-M14/15-Xy1A were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic Xy1A. Measured K(m) and kcat values were closer to those of the other parent, Xy1A. Interestingly, a significant level of heat tolerance up to 60°C, was recorded for FXYN-M3/4-Xy1A in comparison to only 40°C for FXYN-M14/15-Xy1A though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba Xy1A DNA fragment present in FXYN-M3/4-Xy1A were responsible for inducing its thermal stability. (C) 2001 Elsevier Science Inc.

AB - To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and Xy1A of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-Xy1A, FXYN-M9/10-Xy1A, and FXYN-M14/15-Xy1A were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-Xy1A and FXYN-M14/15-Xy1A showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic Xy1A. Measured K(m) and kcat values were closer to those of the other parent, Xy1A. Interestingly, a significant level of heat tolerance up to 60°C, was recorded for FXYN-M3/4-Xy1A in comparison to only 40°C for FXYN-M14/15-Xy1A though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba Xy1A DNA fragment present in FXYN-M3/4-Xy1A were responsible for inducing its thermal stability. (C) 2001 Elsevier Science Inc.

KW - Chimeric xylanases

KW - Family 10 xylanases

KW - Module shuffling

KW - Streptomyces olivaceoviridis

KW - Thermomonospora alba

UR - http://www.scopus.com/inward/record.url?scp=0035793156&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035793156&partnerID=8YFLogxK

U2 - 10.1016/S0141-0229(00)00279-9

DO - 10.1016/S0141-0229(00)00279-9

M3 - Article

VL - 28

SP - 8

EP - 15

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

SN - 0141-0229

IS - 1

ER -