Cloning and expression of a Trichoderma longibrachiatum β-mannanase gene in Pichia pastoris

Abstract

Trichoderma 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 study, a Trichoderma 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, man1 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 preincubation 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 K_m value of 0.95 mg/ml.

Key words: Trichoderma longibrachiatum, mannanase, Pichia pastoris, expression, recombinant.