Cloning and Expression of a NovelTrichoderma virideLaminarinase AI Gene (lamAI)

Abstract

The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens -1,3-glucanase BGN1, but no significant similarity to fungal -1,6-glucanases or -1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo--1,3:1,6-glucan hydrolases as a subclass of endo--1,3-glucanases in some mycoparasitic fungi is suggested.

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

“…They are conserved throughout the GH55 family and also define the origin of exo-glucanase activity observed for SacteLam55A. Although it was previously suggested that some GH55 might have endo-␤-1,3-glucanase activity (21,26,62), sequence conservation in the Ϫ1 and ϩ1 sugar binding subsites and in the "aromatic block" support that most, if not all, GH55s are exo-␤-1,3-glucanases. This conclusion is in accord with biochemical studies carried out on PcLam55A (20).…”

“…Functional Survey of the GH55 Family-To date, the majority of studies on GH55 have been on fungal enzymes (19,20,(22)(23)(24)(25)(26)(27)61). With the exception of a single report on the cloning and sequence analysis of GluA from Arthrobacter sp.…”

“…With the exception of single report on the cloning and sequence analysis of GluA from Arthrobacter sp. NHB-1 (21), previous published work on GH55 has been on fungal members (19,20,(22)(23)(24)(25)(26)(27). This includes the GH55 from Phanerochaete chrysosporium, previously named PcLam55A (20), whose structure has also been determined.…”

Biocatalysts in Control of Phytopatogenic Fungi and Methods for Antifungal Effect Detection

“…The partition coefficients of enzymes laminarinase and chitinase (K o/w ) are greater than 1 indicating affinity of enzymes for microencapsulation in liposomes. However, the mechanisms of microencapsulation are different for each enzyme that may be related with differences of their primary structure and amount of lipophilic nature aminoacids (Nobe et al, 2004). The thermodynamic functions related to the transfer of laminarinase and chitinase from aqueous media to soya lecithin liposomes are summarize in Table 7.…”

Crystal structure and biological implications of a glycoside hydrolase family 55 β-1,3-glucanase from Chaetomium thermophilum

“…Recent genomic sequencing data have showed a number of β-1,3-glucanases genes from the GH55 family in fungi and bacteria and some β-1,3-glucanases of the GH55 family have been cloned, expressed and characterized [6][7][8][9][10][11][12][13][14]. However, the crystal structures of only two β-1,3-glucanases from the GH55 family have been reported so far, one (PcLam55A) from the fungus Phanerochaete chrysosporium [6] and the other (SacteLam55A) from the bacterial Streptomyces sp.…”