Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei

Expression of the Aspergillus aculeatus Endo- beta-1,4-mannanase Encoding Gene ( man1 ) in Saccharomyces cerevisiae and Characterization of the Recombinant Enzyme

The endo-beta-1,4-mannanase encoding gene man1 of Aspergillus aculeatus MRC11624 was amplified from mRNA by polymerase chain reaction using sequence-specific primers designed from the published sequence of man1 from A. aculeatus KSM510. The amplified fragment was cloned and expressed in Saccharomyces cerevisiae under the gene regulation of the alcohol dehydrogenase ( ADH2PT ) and phosphoglycerate kinase ( PGK1PT ) promoters and terminators, respectively. The man1 gene product was designated Man5A. Subsequently, the FUR1 gene of the recombinant yeast strains was disrupted to create autoselective strains: S. cerevisiae Man5 ADH2 and S. cerevisiae Man5 PGK1. The strains secreted 521 nkat/ml and 379 nkat/ml of active Man5A after 96 h of growth in a complex medium. These levels were equivalent to 118 and 86 mg/l of Man5A protein produced, respectively. The properties of the native and recombinant Man5A were investigated and found to be similar. The apparent molecular mass of the recombinant enzyme was 50 kDa compared to 45 kDa of the native enzyme due to glycosylation. The determined Km and V max values were 0.3 mg/ml and 82 mumol/min/mg for the recombinant and 0.15 mg/ml and 180 mumol/min/mg for the native Man5A, respectively. The maximum pH and thermal stability were observed within the range of pH 4-6 and 50degreesC and below. The pH and temperature optima and stability were relatively similar for recombinant and native Man5A. Hydrolysis of an unbranched beta-1,4-linked mannan polymer released mannose, mannobiose, and mannotriose as the main products. Copyright 2001 Academic Press

The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst