expression of a Trichoderma longibrachiatum β-mannanase
gene in Pichia pastoris
J. L. Lim1, 2, F. D. A.
Bakar1, H. M. Yusof 2 and
A. M. A.
1School of Biosciences and
Biotechnology, Faculty of Science and Technology, Universiti
Malaysia, 43600 Bangi, Selangor, Malaysia.
Darby Technology Centre,
1st Floor Block B,
UPM-MTDC III, Lebuh Silikon 43400, Serdang, Selangor,
Tel: +6-03-89215696. Fax: +6-0389252698.
Accepted 11 November, 2011
species are among the primary producers of
β-mannanase, an enzyme that catalyses the hydrolysis of β-1,
4-glycosidic linkages in mannans and heteromannans. In this
species producing high mannanase activity was identified as
Trichoderma longibrachiatum based on sequence
analysis of its rDNA internal transcribed spacer region.
The open reading frame of the gene encoding for
β-mannanase of T. longibrachiatum,
is 1,441 bp and is separated by two introns. The MAN1 amino
acid sequence showed
95% identity to Trichoderma reesei β-mannanase.
Domain analysis classified MAN1 as a member of glycosyl
hydrolase family 5 and detected the presence of a
carbohydrate-binding domain family 1 at its C-terminus. The
recombinant mannanase, rMAN1, was successfully expressed as
a ~60 kDa extracellular recombinant protein in Pichia
pastoris and was verified via western blotting analyses.
The specific activity of the purified rMAN1 was 1416.18
U/mg. The optimal rMAN1 activity was recorded at 55°C and pH
5. The enzyme was stable with 30 min pre-incubation at
temperatures ranging from 4 to 50°C. The enzyme was stable
at pH 4 to 7 but became progressively unstable at pH values
below 3 and above 8. rMAN1 had a high affinity towards
locust bean gum as a substrate, with a Km value
of 0.95 mg/ml.
Trichoderma longibrachiatum, mannanase, Pichia
pastoris, expression, recombinant.