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ABSTRACT: The Clostridium thermocellum cellobiose phosphorylase (CtCBP) is a large protein consisting of 812 amino acids and has great potential in the production of sugar phosphates, novel glycosides, and biofuels. It is relatively stable at 50 °C, but is rapidly inactivated at 70 °C. To stabilize CtCBP at elevated temperatures, two protein-engineering approaches were applied, i.e. site-directed mutagenesis based on structure-guided homology analysis and random mutagenesis at various mutation rates. The former chose substitutions by comparison of the protein sequences of CBP homologs, utilized structural information to identify key amino acid residues responsible for enhanced stability, and then created a few variants accurately. The latter constructed large libraries of random mutants at different mutagenesis frequencies. A novel combinational selection/screening strategy was employed to quickly isolate thermostability-enhanced and active variants. Several stability-enhanced mutants were obtained by both methods. Manually combining the stabilizing mutations identified from both rational and random approaches led to the best mutant (CM3) with the halftime of inactivation at 70 °C extended from 8.3 to 24.6 min. The temperature optimum of CM3 was increased from 60 to 80 °C. These results suggested that a combination of rational design and random mutagenesis could have a solid basis for engineering large proteins.
Molecular BioSystems 04/2012; 8(6):1815-23. · 3.53 Impact Factor
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ABSTRACT: Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).
Applied Microbiology and Biotechnology 06/2011; 92(3):551-60. · 3.42 Impact Factor
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ABSTRACT: Cocktail reception: Biohydrogen is produced in high yield from cellulosic materials and water in a one-pot process catalyzed by up to 14 enzymes and one coenzyme. This assembly of enzymes results in non-natural catabolic pathways. These spontaneous reactions are conducted under modest reaction conditions (32 degrees C and atmospheric pressure).
ChemSusChem 03/2009; 2(2):149-52. · 6.83 Impact Factor
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ABSTRACT: Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and beta-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture.
Methods in molecular biology (Clifton, N.J.) 01/2009; 581:213-31.
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ABSTRACT: Low-cost protein purification methods are in high demand for mass production of low-selling price enzymes that play an important role in the upcoming bioeconomy. A simple protein purification method was developed based on affinity adsorption of a cellulose-binding module-tagged protein on regenerated amorphous cellulose (RAC) followed by modest desorption. The biodegradable cellulosic adsorbent RAC had a very high protein-binding capacity of up to 365mg of protein per gram of RAC. The specifically-bound CBM-protein on the external surface of RAC was eluted efficiently by ethyl glycol or glycerol. This protein separation method can be scaled up easily because it is based on simple solid/liquid unit operations. Five recombinant proteins (CBM-protein), regardless of intercellular or periplasmic form, were purified successfully for demonstration purpose.
Analytica chimica acta 08/2008; 621(2):193-9. · 4.31 Impact Factor
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ABSTRACT: A simple, low-cost, and scalable protein purification method was developed by using a biodegradable regenerated amorphous cellulose (RAC) with a binding capacity of up to 365 mg protein per gram of RAC. The recombinant protein with a cellulose-binding module (CBM) tag can be specifically adsorbed by RAC. In order to avoid using costly protease and simplify purification process, a self-cleavage intein was introduced between CBM and target protein. The cleaved target protein can be liberated from the surface of RAC by intein self-cleavage occurring through a pH change from 8.0 to 6.5. Four recombinant proteins (green fluorescence protein, phosphoglucomutase, cellobiose phosphorylase, and glucan phosphorylase) have been purified successfully.
Journal of Chromatography 07/2008; 1194(2):150-4. · 4.53 Impact Factor
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ABSTRACT: Heterogeneous cellulose accessibility is an important substrate characteristic, but all methods for determining cellulose accessibility to the large-size cellulase molecule have some limitations. Characterization of cellulose accessibility to cellulase (CAC) is vital for better understanding of the enzymatic cellulose hydrolysis mechanism (Zhang and Lynd, Biotechnol. Bioeng. 2004, 88, 797-824; 2006, 94, 888-898). Quantitative determination of cellulose accessibility to cellulase (m2/g of cellulose) was established based on the Langmuir adsorption of the fusion protein containing a cellulose-binding module (CBM) and a green fluorescent protein (GFP). One molecule of the recombinant fusion protein occupied 21.2 cellobiose lattices on the 110 face of bacterial cellulose nanofibers. The CAC values of several cellulosic materials -- regenerated amorphous cellulose (RAC), bacterial microcrystalline cellulose (BMCC), Whatman No. 1 filter paper, fibrous cellulose powder (CF1), and microcrystalline cellulose (Avicel) -- were 41.9, 33.5, 9.76, 4.53, and 2.38 m2/g, respectively. The CAC value of amorphous cellulose made from Avicel was 17.6-fold larger than that of crystalline cellulose - Avicel. Avicel enzymatic hydrolysis proceeded with a transition from substrate excess to substrate limited. The declining hydrolysis rates over conversion are mainly attributed to a combination of substrate consumption and a decrease in substrate reactivity. Declining heterogeneous cellulose reactivity is significantly attributed to a loss of CAC where the easily hydrolyzed cellulose fraction is digested first.
Langmuir 01/2008; 23(25):12535-40. · 4.19 Impact Factor
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ABSTRACT: Presently, it is widely accepted complex systems couldn't be comprehended by studying parts in isolation without examining integrative and emergent properties, and system-level understanding thus has become the focus in biological science. However, it should also be noted that common systematic analysis was restricted to large-scale analysis at a certain level, while the facts that the nature of complex systems is their multi-scale structures was usually neglected or ignored. Therefore, this paper described a multi-scale methodology to investigate the nature of biological complexity and prospected this methodology could lead to a promising revolution in current system-level understanding and the integration of molecular biology databases.
Frontiers in Bioscience 02/2005; 10:961-5. · 3.52 Impact Factor
