A 6-mo-old female Chihuahua was presented with recurrent episodes of hypoglycemia and collapse. Physical examination revealed proportionate dwarfism, retained puppy hair coat, retained deciduous teeth, and open fontanelles. Routine blood tests revealed hypoglycemia, thrombocytosis, hypoproteinemia, and elevated alkaline phosphatase activity. The urinalysis, radiographs, and ultrasonographs were unremarkable. Endocrine testing revealed that insulin-like growth factor 1 was below the detection limit; concentrations of total thyroxine, baseline cortisol, and cortisol stimulated by tetracosactide acetate were within their reference intervals. The pituitary gland showed no organic abnormalities on magnetic resonance imaging. For definitive diagnosis, we conducted the stimulation test for growth hormone (GH) release and diagnosed isolated GH deficiency. Genetic investigation revealed that the present case had 4 point mutations in intronic regions and a 6-bp deletion in exon 5 of GH1. The bioinformatics tool PROVEAN algorithm predicted that the deletion in exon 5 could be deleterious to the function of GH1.

Keywords: dogs; dwarfism; growth hormone deficiency.

The function and application of β-glucosidase attract attention nowadays. β-glucosidase was confirmed of transforming ginsenoside Rb₁ to rare ginsenoside, but the interaction mechanism remains not clear. In this work, β-glucosidase from GH1 family of Paenibacillus polymyxa was selected, and its gene sequence bglB was synthesized by codon. Then, recombinant plasmid was transferred into Escherichia coli BL21 (DE3) and expressed. The UV-visible spectrum showed that ginsenoside Rb₁ decreased the polarity of the corresponding structure of hydrophobic aromatic amino acids (Trp) in β-glucosidase and increased new π-π^* transition. The fluorescence quenching spectrum showed that ginsenoside Rb₁ inhibited intrinsic fluorescence, formed static quenching, reduced the surface hydrophobicity of β-glucosidase, and K_SV was 8.37 × 10³ L/M (298K). Circular dichroism (CD) showed that secondary structure of β-glucosidase was changed by the binding action. Localized surface plasmon resonance (LSPR) showed that β-glucosidase and Rb₁ had strong binding power which KD value was 5.24 × 10^-4 (±2.35 × 10^-5) M. Molecular docking simulation evaluated the binding site, hydrophobic force, hydrogen bond, and key amino acids of β-glucosidase with ginsenoside Rb₁ in the process. Thus, this work could provide basic mechanisms of the binding and interaction between β-glucosidase and ginsenoside Rb₁.

Keywords: ginsenoside Rb1; interaction; molecular docking; multispectral method; β‐glucosidase.

Glycoside hydrolase family 1 (GH1) β-glucosidases (BGLUs) are encoded by a large number of genes and are involved in many developmental processes and stress responses in plants. Due to their importance in plant growth and development, genome-wide analyses have been conducted in the model plant species Arabidopsis thaliana, rice and maize but not in woody plant species, which have important economic and ecological value. In this study, we systematically analyzed Populus BGLUs (PtBGLUs) and demonstrated the involvement of several genes under stress conditions. Forty-four PtBGLUs were identified in Populus databases; these genes were located on 11 chromosomes, and the proteins of several PtBGLU genes were highly similar. More than 90% of PtBGLUs contain three conserved motifs. Collinearity results showed that 44 PtBGLU genes resulted from 12 tandem and 5 segmental duplication events. Phylogenetic analysis revealed that 128 BGLU genes from Populus trichocarpa, A. thaliana and Oryza sativa could be classified into 4 subgroups and subgroup Ⅱ and Ⅳ were differently having PtBGLUs and AtBGLUs. We further investigated whether several PtBGLUs responded to drought stress and ABA treatment, and the results showed that most of the selected BGLU genes were expressed in response to stress, which is consistent with previous studies involving rice and Arabidopsis homologous genes. Large numbers of stress-, hormone-, and development-related elements in the PtBGLU promoters suggest that BGLU genes may be involved in complex networks. Taken together, our results provide valuable information for an improved understanding of β-glucosidase function in woody plants.

Keywords: ABA; Drought; Populus trichocarpa; β-glucosidase.

The nonlinear regression method was used for the evaluation of applicability of the known model equations (Wherland-Gray, Brönsted-Debay-Hukkel and "parallel disks") which describe the ionic strength dependence of the reaction rate between charged molecules to the redox reaction of cytochrome c with sperm whale myoglobin modified at His 12(A10) with the bromoacetate spin label, and pig myoglobin. Unlike the native sperm whale Mb studied earlier [1], the objects chosen have monotonous pH-dependence of the reaction rate and are more simple with regards to electrostatic interactions in the electron-transfer complex. This allowed to study the influence of the total as well as the local protein charge on the correspondence of the ion strength dependencies to the theoretical models and optimal parameters of the equations. It was shown that the models considered, as in the case of the native sperm whale Mb-Cyt c reaction, permit satisfactory description of the experimental data, but the obtained parameters cannot be applied to the whole proteins or their contact sites. In the best case (Wherland-Gray equation) it is possible to do if the distribution of electrostatic potential in the contact area is considered. The reason can be that, unlike other protein redox-systems, the contact sites of both Mb and Cyt c have the charged residues of both signs, and the His GH1 residue located in the contact Mb site is not only involved in the electrostatic interactions in electron-transfer complex, but also participates directly in the mechanism of charge transfer.

Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-β-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-β-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.

The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb's free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the <em>GH1</em>, GH3, GH5, GH9 and <em>GH1</em>0 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

The International Carbohydrate Symposium (ICS), held in Tokyo, included topics covering new developments in the field of glycoscience research. This conference report highlights selected presentations on glycoside hydrolase 1 (GH1) substrate specificity, antibody/lectin binding to oligosaccharides, probing the enzymatic properties of oligosaccharyltransferase, galactolipid biosynthesis, galactose disaccharide binding to a Pseudomonas lectin, O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins, and NMR J-coupling correlations in saccharides.

The reaction mechanism of glycoside hydrolases belonging to family 1 (GH1) of carbohydrate-active enzymes classification, hydrolysing β-O-glycosidic bonds, is well characterised. This family includes several thousands of enzymes with more than 20 different EC numbers depending on the sugar glycone recognised as substrate. Most GH1 β-glycosidases bind their substrates with similar specificity through invariant amino acid residues. Despite extensive studies, the clear identification of the roles played by each of these residues in the recognition of different glycones is not always possible. We demonstrated here that a histidine residue, completely conserved in the active site of the enzymes of this family, interacts with the C2-OH of the substrate in addition to the C3-OH as previously shown by 3 D-structure determination.

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC 50 = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

The influence of cupric ions on the oxidation of sperm whale oxymoglobin and soybean oxyleghemoglobin at pH 4.8--7.5 and 10--40 degrees C has been studied. The stability constants of cupric ions to myoglobin were determined: K1 = 3.4 . 10(5) M(-1) for the more reactive center, and K2 = 2.1 . 10(3) M(-1) for the next 5--7 binding centers. The role of ionization of His-GH1 in the process of oxidation of sperm whale myoglobin is discussed. A mechanism of electron transfer from myoglobin molecule to the external acceptor is presented.

Litopenaeus vannamei (L. vannamei) is one of the most widely cultured shrimp species in the world. The species often suffers from cold stress. To understand the molecular mechanism of cold tolerance, we performed transcriptomic analysis on two contrasting cultivars of L. vannamei, namely, cold-tolerant Guihai 2 (GH2) and cold-sensitive Guihai1 (GH1), under a control temperature (28°C), cold stress (16°C), and recovery to 28°C. A total of 84.5 Gb of sequences were generated from 12 L. vannamei hepatopancreas libraries. The de-novo assembly generated a total of 143,029 unigenes with a mean size of 1,052 bp and an N50 of 2604 bp, of which 34.08% were annotated in the Nr database. We analyzed the differentially expressed genes (DEGs) between nine comparison groups and detected a total of 21,026 DEGs. KEGG pathways, including lysosome, sphingolipid metabolism and nitrogen metabolism, were significantly enriched by DEGs between different temperatures in GH2. Furthermore, eight of the most significantly DEGs under cold stress from the transcriptomic analysis were selected for quantitative real-time PCR (qPCR) validation. Overall, we compared gene expression changes under cold stress in cold-tolerant and cold-sensitive L. vannamei for the first time. The results may further extend our understanding of the cold stress-response mechanism in L. vannamei.

Keywords: Litopenaeus vannamei; Transcriptomic; cold stress; cold-sensitive cultivars; cold-tolerant cultivars.

A β-glucosidase gene (bsbgl1a) from Bacillus sp. CGMCC 1.16541 was expressed in Escherichia coli BL21 and subsequently characterized. The amino acid sequence shared 83.64% identity with β-glucosidase (WP_066390903.1) from Fictibacillus phosphorivorans. The recombinant β-glucosidase (BsBgl1A) had a molecular weight of 52.2 kDa and could hydrolyze cellobiose, cellotriose, cellotetrose, p-nitrophenyl-β-D-glucopyranoside (pNPG), and p-nitrophenyl-β-D-xylopyranoside (pNPX). Optimal activity for BsBgl1A was recorded at 45 °C with a pH between 5.6 and 7.6, and 100% of its activity was maintained after a 24 h incubation between pH 4 and 9. Kinetic characterization revealed an enzymatic turnover (Kcat) of 616 ± 2 s^-1 (with cellobiose) and 3.5 ± 0.1 s^-1 (with p-nitrophenyl-β-D-glucopyranoside). Interestingly, the recombinant enzyme showed cupric ion (Cu²⁺), sodium dodecyl sulfate (SDS) and alcohol tolerance at 10 mM for Cu²⁺ and 10% for both SDS and alcohol. Additionally, BsBgl1A had high tolerance for glucose (Ki = 2095 mM), which is an extremely desirable feature for industrial applications. Following the addition of BsBgl1A (0.05 mg/ml) to a commercial cellulase reaction system, glucose yields from sugarcane bagasse increased 100% after 1 day at 45 °C. This work identifies a Cu²⁺, SDS, alcohol, and glucose tolerant GH1 β-glucosidase with potential applications in the hydrolysis of cellulose for the bioenergy industry.

Keywords: Alcohol; Bacillus sp.; Cu2+; Glucose tolerance; Heterogeneous expression; SDS; β-Glucosidase.

The ginseng polysaccharides GH1 (100, 200 mg.kg-1) iv reduced liver glycogen and increased adenosine-3',5'-cyclic monophosphate (cAMP) level and adenyl cyclase (AC) activity in mice. The action of GH1 was completely antagonized by propranolol, inhibitor of adrenergic beta receptor. The stimulating effect of GH1 on AC activity was significant 2 and 4 h after iv GH1. However, GH1 at concentration of 20-120 mumol.L-1 in vitro showed no manifest effect on AC activity. GH1 stimulated the activities of 3',5'-cyclic-GMP phosphodiesterases (PDE) and calmodulin (CaM) in a dose-dependent manner. It is suggested that the reduction of liver glycogen induced by GH1 resulted from its obvious increase of cAMP which promoted glycogenolysis and decreased glycogenesis.

β-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred μg/mL β-D-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.

β-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred μg/mL β-D-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.

Host plant resistance, an important strategy of integrated pest management, was examined in the American cranberry, Vaccinium macrocarpon Aiton (Ericaceae). Despite the pressure on cranberry growers to reduce pesticide usage, host plant resistance is not used to help manage insect populations. This study measured field population densities of the three most economically important pest insects in Wisconsin, namely, cranberry fruitworm (Acrobasis vaccinii Riley), sparganothis fruitworm (Sparganothis sulfureana Clemens), and blackheaded fireworm (Rhopobota naevana Hübner), in five different cranberry cultivars, i.e., 'Stevens', 'Ben Lear', 'GH1', 'Mullica Queen', and 'HyRed'. Population densities of male moths of all three species were assessed using pheromone traps in beds of the different cranberry cultivars in commercial marshes in central Wisconsin. For each cultivar, damaged cranberries were collected, and the number of damaged berries and the number of larvae feeding within berries were compared among cultivars. More than 99% of larvae collected were cranberry fruitworm. Mullica Queen and Ben Lear had more damaged berries than Stevens or GH1, and had more larvae than GH1. Conversely, fewer adult male sparganothis fruitworm were found in Ben Lear and Mullica Queen beds than in beds of Stevens or GH1. Adult populations of cranberry fruitworm and blackheaded fireworm were not different among cultivars. Our findings provide evidence of different levels of resistance in common cranberry cultivars, which should inform future plantings and breeding programs.

Fungal hydrolysis of ellagitannins produces hexahydroxydiphenic acid, which is considered an intermediate molecule in ellagic acid release. Ellagic acid has important and desirable beneficial health properties. The aim of this work was to identify the effect of different sources of ellagitannins on the efficiency of ellagic acid release by Aspergillus niger. Three strains of A. niger (GH1, PSH and HT4) were assessed for ellagic acid release from different polyphenol sources: cranberry, creosote bush, and pomegranate used as substrate. Polyurethane foam was used as support for solid-state culture in column reactors. Ellagitannase activity was measured for each of the treatments. Ellagic acid was quantified by high performance liquid chromatography. When pomegranate polyphenols were used, a maximum value of ellagic acid (350.21 mg/g) was reached with A. niger HT4 in solid-state culture. The highest amount of ellagitannase (5176.81 U/l) was obtained at 8h of culture when cranberry polyphenols and strain A. niger PSH were used. Results demonstrated the effect of different polyphenol sources and A. niger strains on ellagic acid release. It was observed that the best source for releasing ellagic acid was pomegranate polyphenols and A. niger HT4 strain, which has the ability to degrade these compounds for obtaining a potent bioactive molecule such as ellagic acid.

The influence of small amounts of low-molecular electron acceptor, potassium ferricyanide, 1 to 20% relative to the cytohrome c concentration, on the rate of electron transfer in the sperm whale oxymyoglobin--horse heart cytochrome c and deoxymyoglobin--cytochrome c systems (under aerobic and anaerobic conditions, respectively) was studied. At low ionic strength, the redox reaction rate was found to increase proportionally to the concentration of ferricyanide in both redox systems. The effect depends on pH in the pH range 5-8, increasing sharply at pH < 6. It was shown that the enhancing of electron transfer is caused by the complexing of [Fe(CN)6]3- with cytohrome c in the Lys72 region, where one of the two strong binding sites for this anion is determined by NMR. Both the high ionic strength and the chemical modification of Lys72 residue inhibit this effect at low ionic strength, markedly decreasing the rate of reaction with myoglobin. Under the same conditions, the effect of ferricyanide in the reaction of oxy-Mb with yeast cytohrome c, which is isopotential to animal cytochromes c but possesses trimethylated Lys72, was several times smaller. In turn, the chemical modification of His residues in myoglobin and the complexing of zinc ion to His119(GH1) almost completely inhibit electron transfer in the systems. Thus, electron transfer between the proteins must proceed through the formation of the Mb.[Fe(CN)6]3-.Cyt c ternary complex, the contacting sites being localized in the His119(GH1) region of myoglobin and near Lys72 of cytohrome c. The increased electron transfer rate in the presence of [Fe(CN)6]3- can be explained by that its binding near Lys72, firstly, provides better electrostatic interactions in the electron transfer complex and, besides, decreases significantly (about 2-fold) the tunneling distance between the two hemes (two lengths of 1.7 and 1.2 nm instead of one of 2.9 nm).

The enzymatic hydrolysis of cellulose and lignocellulosic materials is marked by a rate decrease along the reaction time. Cellobiohydrolase slow dissociation from the substrate and its inhibition by the cellobiose produced are relevant factors associated to the rate decrease. In that sense, addition of β-glucosidases to the enzyme cocktails employed in cellulose enzymatic hydrolysis not only produces glucose as final product but also reduces the cellobiohydrolase inhibition by cellobiose. The digestive β-glucosidase GH1 from the fall armyworm Spodoptera frugiperda, hereafter called Sfβgly, containing the mutation L428V showed an increased kcat for cellobiose hydrolysis. In comparison to assays conducted with the wild-type Sfβgly and cellobiohydrolase TrCel7A, the presence of the mutant L428V increased in 5 fold the initial rate of crystalline cellulose hydrolysis and reduced to one quarter the time needed to TrCel7A produce the maximum glucose yield. As our results show that mutant L428V complement the action of TrCel7A, the introduction of the equivalent replacement in β-glucosidases is a promising strategy to reduce costs in the enzymatic hydrolysis of lignocellulosic materials.

Enzymatic degradation of cellulosic waste to generate renewable biofuels has offered an attractive solution to the energy problem. Synergistic hydrolysis of cellulose residues requires the participation of three different types of cellulases - endoglucanases, exoglucanases, and β-glucosidases (Bgl). Our group has been interested in using Bgl of Cellulomonas biazotea in studies designed to investigate cooperative action among different cellulases. We previously have cloned bgl genes encoding Cba and Cba3, which are C. biazotea Bgl isozymes representing two different Bgl families, respectively; specifically, Glycoside Hydrolase Family 3 (GH3) and Glycoside Hydrolase Family 1 (GH1). To gain an understanding of the complexity of Bgl in C. biazotea, we analyzed E. coli clones containing plasmids into which C. biazotea DNA had been inserted; these clones could hydrolyze 4-methylumbelliferyl β-d-glucopyranoside (MUG) supplemented in solid agar media, suggesting they might contain bgl genes. Through restriction analysis and DNA sequencing, two novel bgl genes, designated cba4 and cba5 and encoding Cba4 (484 amino acids) and Cba5 (758 amino acids) were identified. Cba4 and Cba5 appear to be members of GH1 and GH3, respectively. Both Cba4 and Cba5 were concluded to be genuine cellobiases as each was found to enable their E. coli hosts to survive on media in which cellobiose was the sole carbon source. Despite lacking a typical secretory signal sequence, Cba4 and Cba5 are secretory proteins. Although they are isoenzymes, Cba, Cba3, Cba4, and Cba5 were shown to possess distinct substrate specificities. These four Bgl members may play important roles in hydrolyzing a wide variety of β-glucosides including cellobiose and non-cellulosic substrates.

Simultaneous saccharification and fermentation (SSF) of cellulose via engineered Saccharomyces cerevisiae is a sustainable solution to valorize cellulose into fuels and chemicals. In this study, we demonstrate the feasibility of direct conversion of cellulose into ethanol and a biodegradable surfactant, ethyl-β-d-glucoside, via an engineered yeast strain (i.e., strain EJ2) expressing heterologous cellodextrin transporter (CDT-1) and intracellular β-glucosidase (GH1-1) originating from Neurospora crassa. We identified the formation of ethyl-β-d-glucoside in SSF of cellulose by the EJ2 strain owing to transglycosylation activity of GH1-1. The EJ2 strain coproduced 0.34 ± 0.03 g ethanol/g cellulose and 0.06 ± 0.00 g ethyl-β-d-glucoside/g cellulose at a rate of 0.30 ± 0.02 g·L-1 ·h-1 and 0.09 ± 01 g·L-1 ·h-1 , respectively, during the SSF of Avicel PH-101 cellulose, supplemented only with Celluclast 1.5 L. Herein, we report a possible coproduction of a value-added chemical (alkyl-glucosides) during SSF of cellulose exploiting the transglycosylation activity of GH1-1 in engineered S. cerevisiae. This coproduction could have a substantial effect on the overall technoeconomic feasibility of theSSF of cellulose.

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-D-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-D-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-D-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-D-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-D-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.

β-glucosidases catalyze the final step of cellulose hydrolysis and are essential in cellulose degradation. A β-glucosidase gene, cen502, was identified and isolated from a metagenomic library from Bursaphelenchus xylophilus via functional screening. Analyses indicated that cen502 encodes a 465 amino acid polypeptide that contains a catalytic domain belonging to the glycoside hydrolase family 1 (GH1). Cen502 was heterologously expressed, purified, and biochemically characterized. Recombinant Cen502 displayed optimum enzymatic activity at pH 8.0 and 38 °C. The enzyme had highest specific activity to p-nitrophenyl-β-D-glucopyranoside (pNPG; 180.3 U/mg) and had K m and V max values of 2.334 mol/ml and 9.017 μmol/min/mg, respectively. The addition of Fe2+ and Mn2+ significantly increased Cen502 β-glucosidase activity by 60% and 50%, respectively, while 10% and 25% loss of β-glucosidase activity was induced by addition of Pb2+ and K+, respectively. Cen502 exhibited activity against a broad array of substrates, including cellobiose, lactose, salicin, lichenan, laminarin, and sophorose. However, Cen502 displayed a preference for the hydrolysis of β-1,4 glycosidic bonds rather than β-1,3, β-1,6, or β-1,2 bonds. Our results indicate that Cen502 is a novel β-glucosidase derived from bacteria associated with B. xylophilus and may represent a promising target to enhance the efficiency of cellulose bio-degradation in industrial applications.

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher VMAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with β-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (KS) for cellobiose than KS of the parental strain for glucose but also 5-times lower KS than Michaelis-Menten constant (KM) of the extracellular β-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose.