Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity.

A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering.

Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates - glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70°C, respectively. The optimal enzyme ratios of αGP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 ± 4.6mM FDP was synthesized after 7-h reaction. This de novo ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.

An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch.

Myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here, we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD+ supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e., 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale. Biotechnol. Bioeng. 2017;114: 1855-1864. © 2017 Wiley Periodicals, Inc.

Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes, with applications in synthetic biology.

Two natural nicotinamide-based coenzymes (NAD and NADP) are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism, respectively. Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles. This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways. Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering, which is closely related to product-oriented synthetic biology projects. This review focuses on the methodology of nicotinamide-based coenzyme engineering, with its application in improving product yields and decreasing production costs. Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes. Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review. Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering, especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.

Biomanufacturing: history and perspective.

Biomanufacturing is a type of manufacturing that utilizes biological systems (e.g., living microorganisms, resting cells, animal cells, plant cells, tissues, enzymes, or in vitro synthetic (enzymatic) systems) to produce commercially important biomolecules for use in the agricultural, food, material, energy, and pharmaceutical industries. History of biomanufacturing could be classified into the three revolutions in terms of respective product types (mainly), production platforms, and research technologies. Biomanufacturing 1.0 focuses on the production of primary metabolites (e.g., butanol, acetone, ethanol, citric acid) by using mono-culture fermentation; biomanufacturing 2.0 focuses on the production of secondary metabolites (e.g., penicillin, streptomycin) by using a dedicated mutant and aerobic submerged liquid fermentation; and biomanufacturing 3.0 focuses on the production of large-size biomolecules-proteins and enzymes (e.g., erythropoietin, insulin, growth hormone, amylase, DNA polymerase) by using recombinant DNA technology and advanced cell culture. Biomanufacturing 4.0 could focus on new products, for example, human tissues or cells made by regenerative medicine, artificial starch made by in vitro synthetic biosystems, isobutanol fermented by metabolic engineering, and synthetic biology-driven microorganisms, as well as exiting products produced by far better approaches. Biomanufacturing 4.0 would help address some of the most important challenges of humankind, such as food security, energy security and sustainability, water crisis, climate change, health issues, and conflict related to the energy, food, and water nexus.

Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli.

We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3' and 5' overlapping termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning provides a new cloning method with great simplicity and flexibility. Furthermore, this new method can be modified for the preparation of a large-size mutant library for directed evolution in E. coli. Using this method, it is very easy to generate a mutant library with a size of more than 10(7) per 50 µL of the POE-PCR product within 1 day.

Enhancing functional expression of codon-optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons.

Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc.

Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries.

Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+ to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+ were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+ to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm-2 and 0.255 mA cm-2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm-2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges.

The largest obstacle to the cost-competitive production of low-value and high-impact biofuels and biochemicals (called biocommodities) is high production costs catalyzed by microbes due to their inherent weaknesses, such as low product yield, slow reaction rate, high separation cost, intolerance to toxic products, and so on. This predominant whole-cell platform suffers from a mismatch between the primary goal of living microbes - cell proliferation and the desired biomanufacturing goal - desired products (not cell mass most times). In vitro synthetic biosystems consist of numerous enzymes as building bricks, enzyme complexes as building modules, and/or (biomimetic) coenzymes, which are assembled into synthetic enzymatic pathways for implementing complicated bioreactions. They emerge as an alternative solution for accomplishing a desired biotransformation without concerns of cell proliferation, complicated cellular regulation, and side-product formation. In addition to the most important advantage - high product yield, in vitro synthetic biosystems feature several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this perspective review, the general design rules of in vitro synthetic pathways are presented with eight supporting examples: hydrogen, n-butanol, isobutanol, electricity, starch, lactate,1,3-propanediol, and poly-3-hydroxylbutyrate. Also, a detailed economic analysis for enzymatic hydrogen production from carbohydrates is presented to illustrate some advantages of this system and the remaining challenges. Great market potentials will motivate worldwide efforts from multiple disciplines (i.e., chemistry, biology and engineering) to address the remaining obstacles pertaining to cost and stability of enzymes and coenzymes, standardized building parts and modules, biomimetic coenzymes, biosystem optimization, and scale-up, soon.

Transformation of Bacillus subtilis.

Bacillus subtilis has tremendous applications in both academic research and industrial production. However, molecular cloning and transformation of B. subtilis are not as easy as those of Escherichia coli. Here we developed a simple protocol based on super-competent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids generated by prolonged overlap extension-PCR. Super-competent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. This new protocol is simple (e.g., restriction enzyme, phosphatase, and ligase free), fast, and highly efficient (i.e., ~10(7) or ~10(4) transformants per µg of multimeric plasmid or ligated plasmid DNA, respectively). Shuttle vectors for E. coli-B. subtilis are not required.

New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP.

BACKGROUND: The in-depth understanding of the enzymatic hydrolysis of cellulose with heterogeneous morphology (that is, crystalline versus amorphous) may help develop better cellulase cocktail mixtures and biomass pretreatment, wherein cost-effective release of soluble sugars from solid cellulosic materials remains the largest obstacle to the economic viability of second generation biorefineries. RESULTS: In addition to the previously developed non-hydrolytic fusion protein, GC3, containing a green fluorescent protein (GFP) and a family 3 carbohydrate-binding module (CBM3) that can bind both surfaces of amorphous and crystalline celluloses, we developed a new protein probe, CC17, which contained a mono-cherry fluorescent protein (CFP) and a family 17 carbohydrate-binding module (CBM17) that can bind only amorphous cellulose surfaces. Via these two probes, the surface accessibilities of amorphous and crystalline celluloses were determined quantitatively. Our results for the enzymatic hydrolysis of microcrystalline cellulose (Avicel) suggested that: 1) easily accessible amorphous cellulose on the surface of Avicel is preferentially hydrolyzed at the very early period of hydrolysis (that is, several minutes with a cellulose conversion of 2.8%); 2) further hydrolysis of Avicel is a typical layer-by-layer mechanism, that is, amorphous and crystalline cellulose regions were hydrolyzed simultaneously; and 3) most amorphous cellulose within the interior of the Avicel particles cannot be accessed by cellulase. CONCLUSIONS: The crystallinity index (CrI), reflecting a mass-average (three-dimensional) cellulose characteristic, did not represent the key substrate surface (two-dimensional) characteristic related to enzymatic hydrolysis.

A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose.

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.

Bamboo saccharification through cellulose solvent-based biomass pretreatment followed by enzymatic hydrolysis at ultra-low cellulase loadings.

The modified cellulose solvent- (concentrated phosphoric acid) and organic solvent- (95% ethanol) based lignocellulose fractionation (COSLIF) was applied to a naturally-dry moso bamboo sample. The biomass dissolution conditions were 50 degrees C, 1 atm for 60 min. Glucan digestibility was 88.2% at an ultra-low cellulase loading of one filter paper unit per gram of glucan. The overall glucose and xylose yields were 86.0% and 82.6%, respectively. COSLIF efficiently destructed bamboo's fibril structure, resulting in a approximately 33-fold increase in cellulose accessibility to cellulase (CAC) from 0.27 to 9.14 m(2) per gram of biomass. Cost analysis indicated that a 15-fold decrease in use of costly cellulase would be of importance to decrease overall costs of biomass saccharification when cellulase costs are higher than $0.15 per gallon of cellulosic ethanol.

Fractionating recalcitrant lignocellulose at modest reaction conditions.

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (50 degrees C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ( approximately 97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of beta-glucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.

Enzyme-microbe synergy during cellulose hydrolysis by Clostridium thermocellum.

Specific cellulose hydrolysis rates (g of cellulose/g of cellulase per h) were shown to be substantially higher (2.7- to 4.7-fold) for growing cultures of Clostridium thermocellum as compared with purified cellulase preparations from this organism in controlled experiments involving both batch and continuous cultures. This "enzyme-microbe synergy" requires the presence of metabolically active cellulolytic microbes, is not explained by removal of hydrolysis products from the bulk fermentation broth, and appears due to surface phenomena involving adherent cellulolytic microorganisms. Results support the desirability of biotechnological processes featuring microbial conversion of cellulosic biomass to ethanol (or other products) in the absence of added saccharolytic enzymes.

Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation.

The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated for cellulose-grown chemostat cultures, corresponding to n = 4.20 +/- 0.46. Determination of intracellular beta-glucan radioactivity resulting from 14C-labeled substrates showed that uptake is different for cellulose and cellobiose (G2). For 14C-cellobiose, radioactivity was greatest for G2; substantially smaller but measurable for G1, G3, and G4; undetectable for G5 and G6; and n was approximately 2. For 14C-cellulose, radioactivity was greatest for G5; lower but substantial for G6, G2, and G1; very low for G3 and G4; and n was approximately 4. These results indicate that: (i) C. thermocellum hydrolyzes cellulose by a different mode of action from the classical mechanism involving solubilization by cellobiohydrolase; (ii) bioenergetic benefits specific to growth on cellulose are realized, resulting from the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic cleavage of beta-glucosidic bonds; and (iii) these benefits exceed the bioenergetic cost of cellulase synthesis, supporting the feasibility of anaerobic biotechnological processing of cellulosic biomass without added saccharolytic enzymes.

Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum.

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.

Kinetics and relative importance of phosphorolytic and hydrolytic cleavage of cellodextrins and cellobiose in cell extracts of Clostridium thermocellum.

Rates of phosphorolytic cleavage of beta-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60 degrees C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35 degrees C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by > or = 20-fold for both cellobiose and cellopentaose over a 10-fold range of beta-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of beta-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V(max) values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K(m) for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).