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

Abstract

bIdentification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel ␤-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a ␤-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G 4 S) 3 -Gluc1C, demonstrated 3.2-and 2-fold higher molar specific activities for ␤-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.

Diversity and functional significance of cellulolytic microbes living in termite, pill-bug and stem-borer guts

“…In previous studies we showed that Paenibacillus ICGEB2008 isolated from the gut of the cotton bollworm produced several biomass-degrading enzymes, including cellulases and hemicellulases1819. Therefore, we tested the expression levels in the gut microbes of major glycosyl hydrolase enzymes belonging to the categories of cellulase, hemicellulase, mannanase and glucanase.…”

“…Hydrolytic enzymes have been reported in both the extracellular and cell-bound fractions18192021. More often, the enzymes hydrolysing amorphous polysaccharides are secreted, while those hydrolysing crystalline substrates are found in the membrane-bound fraction of the bacteria1820.…”

“…More often, the enzymes hydrolysing amorphous polysaccharides are secreted, while those hydrolysing crystalline substrates are found in the membrane-bound fraction of the bacteria1820. Enzymes hydrolysing dimeric sugars into monomeric forms can be found in both fractions1922. Therefore, we selected substrates to analyse the hydrolysing enzymes present in both the extracellular and cell-bound fractions.…”

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

“…Compared to BglB, the BglB-Y301H mutant had reduced T m (40.5°C versus 47°C) (Fig. 5B and C) (36,37) and optimal temperature (35°C versus 45°C) (Fig. 4B).…”

Assembly of cellulases with synthetic protein scaffolds in vitro

“…There have been various attempts to produce multienzyme assemblies in vitro, including by gene fusion, protein or DNA scaffold construction, and chemical modification [3]. Although the simplest way is by enzyme fusion, this often results in loss of enzymatic activity or formation of inclusion bodies [4], while chemical modification can also impair enzymatic activity [5]. Moreover, the high cost of generating DNA scaffolds makes this approach infeasible on a large scale [6,7].…”