Production of cellulosic ethanol in Saccharomyces cerevisiae heterologous expressing Clostridium thermocellum endoglucanase and Saccharomycopsis fibuligera β-glucosidase genes

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and β-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an α-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and β-glucosidase was able to produce ethanol from β-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.     

Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding β-glucosidase activity

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in a Lac⁻mutant of Escherichia coli HB101. Five clones expressing β-glucosidase activity were shown by restriction enzyme analysis to contain a common 4.4 kbp fragment of inserted DNA. Hybridization of recombinant plasmids with chromosomal DNA ratified the physical maps of the inserted DNA and was further used to confirm that the 4.4 kbp fragment was common to all five clones. Enzyme activity, comprising cellobiase and aryl-β-glucosidase, was similar with respect to substrate specificity for each of the five clones, and was expressed independently of the orientation of the cloned DNA. A differential effect of temperature on activity of the cellobiase and aryl-β-glucosidase activities was observed but in other respects, the properties of the cloned β-glucosidase corresponded to those of the single β-glucosidase previously described for C. thermocellum.     

Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning,characterization and enhanced thermostability

ORF Cthe0357 from the thermophilic bacterium Clostridium thermocellum ATCC 27405 that encodes a putative α-glucan phosphorylase (αGP) was cloned and expressed in Escherichia coli. The protein with a C-terminal His-tag was purified by Ni²⁺ affinity chromatography; the tag-free protein obtained from a cellulose-binding module–intein–αGP fusion protein was purified through affinity adsorption on amorphous cellulose followed by intein self-cleavage. Both purified enzymes had molecular weights of ca. 81,000 and similar specific activities. The optimal conditions were pH 6.0–6.5 and 60 °C for the synthesis direction and pH 7.0–7.5 and 80 °C for the degradation direction. This enzyme had broad substrate specificities for different chain length dextrins and soluble starch. The thermal inactivation of this enzyme strongly depended on temperature, protein concentration, and certain addictives that were shown previously to benefit the protein thermostability. The half lifetime of 0.05 mg αGP/mL at 50 °C was extended by 45-fold to 90 h through a combined addition of 0.1 mM Mg²⁺, 5 mM DTT, 1% NaCl, 0.1% Triton X-100, and 1 mg/mL BSA. The enzyme with prolonged stability would work as a building block for cell-free synthetic enzymatic pathway biotransformations, which can implement complicated biocatalysis through assembly of a number of enzymes and coenzymes.     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

A theoretical framework for β-glucan degradation during barley malting

During malting, barley germinates and produces hydrolytic enzymes that de-structure the endosperm, making the grains soft and friable. This process starts close to the embryo and spreads throughout the whole grain. It is leaded by the degradation of cell walls, which are mainly constituted of β-glucans. Fast and extended breakdown of β-glucans occurs by means of an expanding reaction front driven by β-glucanase, and appears to follow pseudo-first-order kinetics. Endosperm permeabilization to macromolecules is closely linked to the dismantling of cell walls, thus that access to β-glucans by β-glucanase itself is limited. It is shown that the kinetics of β-glucan degradation during malting are consequent to this condition, and can be explained according to an anomalous evolution of the reverse quasi-steady-state approximation (rQSSA) for enzymatic reactions. In fact, kinetics based on the rQSSA include a transient phase wherein fast substrate depletion is indeed of pseudo-first-order. In the germinating barley, the conditions in which the physical modification of the endosperm occurs are shown to be suitable for the fast transient to persist in dynamic equilibrium while it progressively expands throughout the grain, depleting most β-glucans and, then, establishing the overall kinetics of β-glucan breakdown.     

Degradation of β-lactam antibiotics by polyacrylamide-entrapped β-lactamase-producingE. coli cells

Summary Cells of a -lactamase producingE. coli strain were immobilized with acrylamide and lyophilized. The gel particles containing the entrapped cells were used like an immobilized enzyme to study the inactivation of -lactam antibiotics. The substrate profile of the -lactamase was determined and the action of -lactamase inhibitors studied.     

Physiological activities of a β-glucan produced by Panebacillus polymyxa

In vitro bioactivities of a beta-glucan produced by Panebacillus polymyxa JB115 were investigated. Nitric oxide production by RAW 264.7 macrophage cells pre-treated with beta-glucan JB115 (from 0.1 to 1 mg ml(-1)) was significantly increased, compared to that in untreated cells (P < 0.001). The beta-glucan JB115 increased superoxide radical-scavenging activity by 66% at 1 mg ml(-1). It also suppressed hyaluronidase (32%) and collagenase (33%) activities and, additionally, displayed antitumor activity, blocking the growth of Sarcoma 180 cells in a concentration-dependent manner. The immune-stimulatory, antioxidant, collagenase inhibitory and hyaluronidase inhibitory effects of the beta-glucan support its potential role in the prevention of bacterial disease against fish and in the protection of skin against aging.     

Enzymatic fingerprinting of arabinoxylan and β-glucan in triticale, barley and tritordeum grains

Enzymatic fingerprinting of arabinoxylan (AX) and β-glucan using endo-xylanase and lichenase, respectively, helps determine the structural heterogeneity between different cereals and within genotypes of the same cereal. This study characterised the structural features of AX and β-glucan in whole grains of eight triticale cultivars grown at two locations, 20 barley cultivars/lines with wide variation in composition and morphology and five tritordeum breeding lines. Principal component analysis (PCA) resulted in clear clustering of these cereals. In general, barley and tritordeum had a higher relative proportion of highly branched arabinoxylan oligosaccharides (AXOS) than triticale. Subsequent analysis of triticale revealed two clusters based on growing region along principal component (PC) 1, while PC2 explained the genetic variability and was based on mono-substitution and di-substitution in AX fragments. PCA of β-glucan features separated the three cereals based on β-glucan content. The molar ratio of trisaccharide to tetrasaccharide was 2.5-3.4 in triticale, 2.3-3.3 in barley and 2.8-3.4 in tritordeum. Barley showed a strong positive correlation (r=0.86) between β-glucan content and relative proportion of trisaccharide. The results show that structural features of AX and β-glucan vary between and within triticale, barley and tritordeum grains which might be important determinants of end-use quality of grains.     

Molecular cloning ofClostridium thermocellum genes involved in β-glucan degradation in bacteriophage lambda

A genomic library ofClostridium thermocellum DSM 1237 was constructed in a bacteriophage lambda vector and screened for hydrolysis of carboxymethyl cellulose (CMC), lichenan and methylumbelliferyl--glucoside. Recombinant clones expressing four -glucanases and two distinct -glucosidases were obtained. The -glucanase activities could be classified with respect to their substrate specificities as -1, 4-endoglucanase (CMCase), -1, 3-endoglucanase (laminarinase), and -1, 3-1, 4-endoglucanases (lichenanase). The -glucosidases were identified as cellobiases hydrolyzing both aryl--glucosides and cellobiose.     

Structural analyses and yeast production of the β-1,3-1,4-glucanase catalytic module encoded by the licB gene of Clostridium thermocellum

A thermophilic glycoside hydrolase family 16 (GH16) β-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 Å resolution. The enzyme folds into a classic GH16 β-jellyroll architecture which consists of two β-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner β-sheet. Two Bis–Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 β-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris, encouraging its further commercial applications.     

Characterization of two β-glucosidase genes fromClostridium thermocellum

Summary Two -glucosidase genes, designatedbglA andbglB, were isolated from a gene bank ofClostridium thermocellum DSM 1237. The coding sequences forbglA andbglB were located on non-homologous DNA fragments of 3.2– and 3.4-kb, respectively. Both genes direct inEscherichia coli the synthesis of cytoplasmic -glucosidases, which differ with respect to substrate specificity and temperature profile. The properties of thebglA-encoded -glucosidase A closely resemble that of a -glucosidase previously isolated fromC. thermocellum cultures.     

Simultaneous overproduction of extracellular endoglucanase and intracellular β-glucosidase by genetically engineered Bacillus subtilis

Summary Simultaneous overproduction of intracellular -glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the -glucosidase-endoglucanase gene(E) or the endoglucanase--glucosidase gene(E) under the control of a strong engineered promoter, BJ27U88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the -glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.     

Degradation of β-Conglycinin β-Homotrimer by Papain: Independent Occurrence of Limited and Extensive Proteolysis

Limited and extensive proteolysis occur when β-conglycinin β homo-trimer (β₃-conglycinin) from soybeans is attacked by papain. Slow limited proteolysis is restricted to cleavage of β₃-conglycinin polypeptides into subunit halves (N- and C-terminal domains) that are further slightly truncated. The kinetics of limited and extensive proteolyses analyzed separately indicates that the two processes occur independently from the very beginning of the reaction. In contrast, limited proteolysis of phaseolin from common beans has been found to be prerequisite for the onset of its extensive proteolysis. The dramatic distinction between the degradation patterns of β₃-conglycinin and phaseolin, homologous storage 7S globulins, suggests the existence of intrinsic differences in their structures. This hypothesis is supported by comparative analysis of the accessibilities to the solvent of amino acid residues in phaseolin and β₃-conglycinin structures, which indicated the relatively low packing density of the latter, resulting in enhanced susceptibility of it to extensive proteolysis.     

A 13C CP/MAS NMR spectroscopy and AFM study of the structure of Glucagel™, a gelling β-glucan from barley

The structure of Glucagel™, a mixed-linked (1→3), (1→4)-β-d-glucan extracted from barley, was examined using ¹³C CP/MAS NMR spectroscopy and atomic force microscopy (AFM). Results from ¹³C CP/MAS NMR spectroscopy showed that Glucagel™ contained regions with two distinct conformations. In some of the regions the β-glucan chains associated to form a unique conformation, the A-conformation, while in the other regions the β-glucan chains were in an amorphous conformation. Dilute solutions of Glucagel™ were prepared for imaging by dissolving Glucagel™ in water at 90 °C. If the dilute solution was immediately deposited onto mica and the surface dried, then no fine detail was seen in the AFM image. However, when dilute solutions of Glucagel™ were left for several days before being deposited onto the mica surface, individual fibres could be clearly imaged. These results suggested that in gels formed from Glucagel™, junction zones occur because of the interaction of two β-glucan chains in the A-conformation.     

Purification and characterization of an endoglucanase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> highly active against barley β-glucan and xyloglucan

An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan, xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN₁ following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg⁻¹ protein⁻¹ against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated in the presence of Cu²⁺, Mg²⁺, Ca²⁺, Na⁺, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K _cat) and catalytic efficiency (K _cat/km) of 19.11 × 10⁵ min⁻¹ and 29.7 × 10⁵ mM⁻¹ min⁻¹ against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose, cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides.     

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants K _a ～ 1.2·10⁵ and 3.0·10⁵ M^?1 with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with K _a of 5.0·10^?5 M^?1. Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of K _a.