Construction of the bifunctional enzyme cellulase-β-glucosidase from the hyperthermophilic bacterium Thermotoga maritima

Abstract

An artificial bifunctional enzyme, cellulase-beta-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and beta-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or beta-glucosidase. The fusion enzyme was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70 degrees C over 30 min, and optimal beta-glucosidase activity at pH 7.0 and 80 degrees C over 30 min.

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

“…It has often been observed previously that the fusion of two or more catalytic domains leads to reduction in catalytic activity of one or both of the domains (2,18,19). We, therefore, decided to construct six fusion proteins, each containing a catalytic domain of Endo5A and Gluc1C, as mentioned in Materials and Methods.…”

“…This prompted us to construct a bifunctional chimera based on Endo5A and Gluc1C. Two such kinds of chimeras have been reported in the literature (18,23). In one case, a fusion of endoglucanase and ␤-glucosidase from Thermotoga maritima was made and reported to have both activities, albeit they were 70% less than those of the individual enzymes (18).…”

“…Single polypeptides carrying more than one cellulolytic activity that have either been isolated from nature (12,29) or constructed in the laboratory (2,11,18,19,23,24) would certainly have advantages as candidate enzymes in the cellulase consortium.…”

Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

“…Common strategies to alter proteins include site-directed mutagenesis and directed evolution (e.g., error-prone PCR). However, site-directed mutagenesis requires a clear understanding of the relationship between enzyme structure and function, and directed evolution requires a straightforward and efficient high-throughput screening method (3)(4)(5).…”

Multimeric Hemicellulases Facilitate Biomass Conversion

“…We and others have demonstrated that the creation of artificial, multifunctional, lignocellulosic hydrolases is a realistic and practical approach for the improvement of biomass conversion (5,8,9,12). These novel enzymes can be used alone, when a 1:1:1 stoichiometry is preferred, or in mixtures with small quantities of other enzymes to create an optimal enzyme cocktail when enzyme ratio FIG.…”