Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovoranscellulose binding domain and its application in hydrolysis of microcrystalline cellulose

Abstract

BackgroundThe complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation.ResultsThe glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V max /K m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188.ConclusionsThe hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

“…However, the specific catalytic activity of Bgl1 was reduced by fusion to the C-terminus, but slightly increased when produced as the N-terminal fusion partner enzyme. Shifts in pH and temperature optima by fusion of protein candidates were also described in other examples (Fan et al 2009b ; Ribeiro et al 2011 ; Zhao et al 2013 ).…”

Extraction and characterization of cellulose microfibers from Retama raetam stems

“…As a result, nowadays, the subject of many researchers worldwide focuses on the need to find alternative fiber sources [4] . Among all natural fibers, cellulose has attracted much interest as it is the most abundant renewable resource in nature and the degradation of cellulosic biomass is an important part of the biosphere's carbon cycle [5] . Its existence as the common material of plant cell walls was first investigated by Braconnot in 1819 [6] and Payen in 1838 [7] .…”

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass

“…Gene fusion is an essential tool in systems and synthetic biology, and has been widely used in the production of synthetic bifunctional enzymes [24][25][26][27]. In cellulose hydrolysis, gene fusion has been used in studying functions and effectiveness of various CBMs [28][29][30] as well as to create enzymes with multiple activities [31][32][33].…”