Production Of Beta-glucosidase Research Articles

Selection of optimal culture conditions for expression of recombinant beta-glucosidase in Escherichia coli

Beta-glucosidase (BGL) is an enzyme involved in the hydrolysis of cellulose and plays an important role in many biological processes. This enzyme is widely available in animals, plants, and microorganisms. Expression of the recombinant enzyme in Escherichia coli is considered a suitable choice for high production via a fast growth rate and feasible manipulation. In this study, we optimised some conditions for the high soluble production of recombinant beta-glucosidase whose gene sequence was mined from the DNA metagenome of Cuc Phuong humus surrounding white rot fungi. The gene bgl was cloned in pET 22b(+) and expressed in the E. coli Rosetta 1 strain. Production of the recombinant BGL was examined at temperature ranges from 18 to 37oC. The recombinant BGL was obtained as a soluble form at low temperatures, and the optimal temperature was 25°C. In comparison to LB, TB, SB, PE media, mTB rich medium in presence of glucose produced the most BGL. Besides, the results assessing the inducer condition showed that the best IPTG concentration for producing the BGL was 0.3 mM IPTG. Furthermore, some fermentation conditions affecting the level of BGL production were assessed as the induction point and harvesting time. The BGL production was the most suitable when the cell at mid-log phase with an OD600 1 and harvesting time after 4 hours of induction. Importantly, the recombinant BGL had good activity on the esculin substrate plate. Based on the selected conditions, the recombinant BGL could be produced high amount to facilitate for future purification and characterization of the recombinant BGL.

Optimization of hydrolases production from cassava peels by Trametes polyzona BKW001

The saccharification of biomass with hydrolases from fungi is considered as an efficient and sustainable approach to obtain fermentable sugars. The expression of these hydrolytic enzymes by fungi is mainly by a substrate-induced mechanism, which depends on prevailing environmental conditions. Trametes polyzona, a white rot fungus, is regarded as one of the most efficient producers of hydrolases for the hydrolysis of biomass. This study sought to examine and optimize cultivation conditions for the efficient production of hydrolases by Trametes polyzona (T. polyzona). Hydrolase production by T. polyzona was examined using parametric optimization. Crude enzyme extracts obtained under varying cultivation conditions were profiled for endoglucanase, betaglucosidase, exoglucanase, xylanase and amylase activities to determine the optimal conditions for hydrolase production. Significantly (p ≤ 0.05) higher endoglucanase (1.97 ± 0.04 U/mL), xylanase (0.87 ± 0.05 U/mL) and amylase (1.16 ± 0.27 U/mL) activities were achieved by the solid-state cultivation in comparison to the submerged cultivation of T. polyzona. Maximum production of endoglucanase, betaglucosidase, exoglucanase, xylanase and amylase were recorded at 14 days of cultivation on cassava peels, 60% moisture level, temperature 30°C, pH 6, and supplementation with 0.3% w/w urea and 0.03% w/w Mg2+. A solid to liquid ratio of 1:2 was found to be ideal for the solid-liquid extraction of hydrolases. This study has proven that optimization of cultivation conditions is an efficient approach to enhance hydrolases production by T. polyzona BKW001. The hydrolase profile of the enzyme cocktail suggests a potential suitability and adoption for ethanol biorefinery from lignocellulosic biomass.

Production, immobilization and characterization of beta-glucosidase for application in cellulose degradation from a novel Aspergillus versicolor

Beta-glucosidase (EC 3.2.1.21) catalyzes the hydrolysis of cellobiose and cellooligosaccharides containing (1 → 4)-beta-glycosidic bonds to glucose, which is crucial in cellulosic ethanol production. In this study, Aspergillus versicolor, a novel highly productive beta-glucosidase strain, was first isolated from Camptotheca acuminata seeds. The highest beta-glucosidase activity with 812.86 U/mL was obtained by using the response surface methodology, and a 14.4-fold has increased compared to the control. The beta-glucosidase was then purified to homogeneity with recovery yield and specific activity of 25.98% and 499.15 U/mg, respectively. To enhance its stability and recyclability, the purified beta-glucosidase was first immobilized onto magnetic MnO2 by electrostatic adsorption. The immobilized materials were characterized by FR-IT, TEM and FE-SEM. Compared with the free beta-glucosidase, the immobilized enzyme exhibited enhanced thermal stability (1.5-fold raise in half-life at 50 °C), and reusability (holding over 60% activity after eight cycles), besides, the optimum pH has increased to 6.0. Substrate specificity research suggested that the enzyme had high hydrolytic activity on cellobiose. It also had a hydrolysis effect on (1 → 3) and (1 → 6)-beta-glycosidic linkages. Application trials in cellulose hydrolysis revealed that the immobilized enzyme was comparatively more effective. Our results suggested this novel immobilized beta-glucosidase makes a promising alternative for the cellulosic ethanol production.

Enhancement, production, and immobilization of beta-glucosidase from Zobellella denitrificans VIT SB117 and its utilization in bioethanol production from lignocellulosic feedstock

β-Glucosidase from novel bacteria Zobellella denitrificans VIT SB117 was isolated, and to increase the production of the enzyme, various growth parameters of the bacteria were optimized. Plackett–Burman design and response surface methodology helped determine the most significant parameters (fructose, temperature, and culture volume) resulting in a 10-fold increase in enzyme activity. The enzyme was purified and kinetics study for free and immobilized enzyme revealed Km of 4.76 mM and 8.39 mM, Kcat of 255.02 s−1 and 114.02 s−1, and Vmax of 4.33 mg/s and 1.25 mg/s, respectively. Enzyme characterization determined optimum substrate concentration and incubation time as 3.5 mM and 10 min respectively for the free enzyme, and 4 mM and 20 min respectively for the immobilized enzyme for maximum activity. pH 5, 45 °C incubation temperature and addition of Mg2+ and Mn2+ ions exhibited similar stimulatory effects on free and immobilized enzyme activities while Hg2+ ions showed strong inhibitory effects. The immobilized enzyme had negligible loss of activity after a month’s storage at 2–4 °C in acetate buffer and ~ 27.76% residual activity after 17 continuous cycles. These optimized parameters were employed for bioethanol production from lignocellulosic wastes. A total cellulose recovery of 52.77% was achieved after pretreatment. Release of ~ 57 mg/g substrate reducing sugars was achieved by enzymatic hydrolysis using immobilized cellulase enzyme complex that produced ~ 5.46 mg/ml bioethanol after 144 h of fermentation using yeast.

Evaluation of agricultural byproducts for the production of betaglucosidase by Aspergillus niger MBT-2 using solid state fermentation

The experiment was conducted to isolate and screen fungal strain and optimization of solid-state fermentation conditions for enhanced production of β-glucosidase. Different fungal cultures were isolated and screened for β-glucosidase production. The physicochemical and nutritional parameters were optimized for enhanced production of β-glucosidase from higher producer. Among all the isolates the isolate which exhibited highest β-glucosidase potential was identified and assigned the code as Aspergillus niger MBT-2. The optimum β-glucosidase production was obtained in M5 medium containing wheat bran after 72 hrs of incubation at 40°C, pH 6 and 20 ml of moisture contents. In addition to this 2% fructose and 2% yeast extract proved to be best carbon and nitrogen sources, respectively and gave maximal enzyme productivity. The exploitation of agricultural by products as a substrate reduced the production cost of enzyme and makes the process economical. The Aspergillus niger MBT-2 has promising potential of bioconversion of low-cost material into valuable product like β-glucosidase.

Beta-Glucosidase Production Optimization from Newly Isolated Aspergillus tubingensis IMMIS2 Using Taguchi Statistical Design

Fungal production of beta-glucosidase was optimized by Taguchi statistical method using corn stover as substrate. Taguchi statistical design depicted the highest glucosidase activity (116 μg mL−1 min−1) by Aspergillus tubingensis IMMIS2 using 5 g of 80-mm mesh-sized substrate with 50% moisture contents, 0.1 g of urea, 0.2 g of KCl, 0.3 g of CaCl2, 0.01 g of MgSO4 and 3 mL of the fungal culture as inoculum. Main effect plot for SN ratio exposed that some parameters (corn stover as substrate, corn stover particle size, urea level and CaCl2) had significant effect on cellulase production as compared to some other parameters (inoculum size, KCl and MgSO4 levels). Our findings of the present study will be helpful for cost-effective and optimum production of commercially important enzymes using agro-industrial wastes as growth substrates.

RETRACTED: Genetic modification: A tool for enhancing beta-glucosidase production for biofuel application

Beta-glucosidase (BGL) is a rate-limiting enzyme for cellulose hydrolysis as it acts in the final step of lignocellulosic biomass conversion to convert cellobiose into glucose, the final end product. Most of the fungal strains used for cellulase production are deficient in BGL hence BGL is supplemented into cellulases to have an efficient biomass conversion. Genetic engineering has enabled strain modification to produce BGL optimally with desired properties to be employed for biofuel applications. It has been cloned either directly into the host strains lacking BGL or into another expression system, to be overexpressed so as to be blended into BGL deficient cellulases. In this article, role of genetic engineering to overcome BGL limitations in the cellulase cocktail and its significance for biofuel applications has been critically reviewed.

Transcriptional analysis of genes encoding β-glucosidase of Schizophyllum commune KUC9397 under optimal conditions.

The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

Effect of pH, Temperature, and Chemicals on the Endoglucanases and β-Glucosidases from the Thermophilic Fungus Myceliophthora heterothallica F.2.1.4. Obtained by Solid-State and Submerged Cultivation.

This work reports endoglucanase and beta-glucosidase production by the thermophilic fungus Myceliophthora heterothallica in solid-state (SSC) and submerged (SmC) cultivation. Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmC. Highest endoglucanase production in SSC occurred after 192 hours: 1,170.6 ± 0.8 U/g, and in SmC after 168 hours: 2,642 ± 561 U/g. The endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimal pH and temperature. The number of endoglucanases was also different: six isoforms in SSC and ten in SmC. Endoglucanase activity remained above 50% after incubation between pH 3.0 and 9.0 for 24 h for both cultivation systems. The effect of several chemicals displayed variation between SSC and SmC isoenzymes. Manganese activated the enzymes from SmC but inhibited those from SSC. For β-glucosidases, maximum production on SmC was 244 ± 48 U/g after 168 hours using cardboard as carbon source. In SSC maximum production reached 10.9 ± 0.3 U/g after 240 h with 1 : 1 wheat bran and sugarcane bagasse. Manganese exerted a significant activation on SSC β-glucosidases, and glucose inhibited the enzymes from both cultivation systems. FeCl3 exerted the strongest inhibition for endoglucanases and β-glucosidases.

Screening and characterization of beta-glucosidase production by Saccharomyces cerevisiae

Beta-Glucosidases (BGS) are, the group of hydrolase enzymes, involved in the degradation processes and many biological processes. Due to demand, intensive screening of BGS is required to explore the natural microbial source of BGS. The current study deals with isolation and identification of BGS producing S. cerevisiae from Thai fruits & beverages and assessment of impact of pH, temperature, and salt concentration on BGS production. About 34 samples were collected. Yeast cells were isolated by plate method and characterized. About ten different strains were isolated and identified. The strain has been confirmed as S. cerevisiae through ribosomal sequencing. The optimization of BGS production was achieved by Box-Behnken design and Response Surface Methodology and confirmed that pH 4.0, temperature at 40 C, and 0.5% of NaCl are optimum conditions. The kinetic analysis suggested that 24 h of incubation achieve the maximum yield. The reported S. cerevisiae strain could be the safer source for BGS. Further studies on enzyme recovery and purification will unbolt the way to attain high-quality microbial enzyme.

Screening of carbon sources for beta-glucosidase production by Aspergillus saccharolyticus

The fungus Aspergillus saccharolyticus was found to produce a culture broth rich in beta-glucosidase activity, an enzyme which plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct application of fungal fermentation broths produced on-site in a biorefinery may be an integral part of a biorefinery for lowering the cost associated with the use of commercial enzymes for saccharification of biomass. Utilization of low value slip streams from the biorefinery as substrates for such an on-site enzyme production would be ideal to reduce costs. In order to understand which carbon sources that support growth and trigger A. saccharolyticus to produce beta-glucosidases, carbon sources, ranging from monomer sugars to complex lignocellulosic biomasses, including pretreated and hydrolyzed corn stover fractions, were investigated as substrates and inducers of enzyme production. A convenient micro titer plate experimental setup was developed that facilitated a fast screening for beta-glucosidase activity on the different carbon sources. The greatest beta-glucosidase activity was found when A. saccharolyticus was cultivated on media containing xylose, xylan, wheat bran, and pretreated corn stover. In a refinery, beta-glucosidase production by A. saccharolyticus could with success be based on the biomass hemicelluloses and their degradation products which cannot be converted by conventional yeast.

Production, purification and characterization of novel beta glucosidase from newly isolated Penicillium simplicissimum H-11 in submerged fermentation.

β-Glucosidase is an important component of the cellulase complex. It not only hydrolyzes cellobiose and short-chain cellooligosaccharides to glucose, but also removes the inhibitory effect of cellobiose on the β-1, 4-endoglucanase and exoglucanase, thereby increasing the overall rate of cellulose biodegradation. β-glucosidasefrom culture supernatant of a fungus Penicillium simplicissimum was purified to homogeneity, by using ammonium sulfate fraction, Sephadex G-100 chromatography, and its properties were studied. The molecular mass of the enzyme was about 126.0 kDa, as identified by 12% SDS-PAGE. The optimum pH and temperature were 4.4 ~ 5.2 and 60 °C, respectively. The enzyme was stable in pH 5.2 ~ 6.4 and under 40 °C. Metal profile of the enzyme showed that Mn(2+) enhances its activity, while Cu(2+), Co(2+)and Fe(3+) cause obvious inhibition. The K m and V max was 14.881 mg/ml and 0.364 mg ml/min against salicin as a Substrate. This enzyme had secondary protein structure as evidenced by FTIR spectrum.

Optimization of β-Glucosidase Production by a Strain of Stereum hirsutum and Its Application in Enzymatic Saccharification

A high beta-glucosidase (BGL)-producing strain, Stereum hirsutum, was identified and isolated and showed a maximum BGL activity (10.4 U/ml) when cultured with Avicel and tryptone as the carbon and nitrogen sources, respectively. In comparison with other BGLs, BGL obtained from S. hirsutum showed a higher level of activity to cellobiose (V(max) = 172 U/mg, and k(cat) = 281/s). Under the optimum conditions (600 rpm, 30°C, and pH 6.0), the maximum BGL activity of 10.4 U/ml with the overall productivity of 74.5 U/l/h was observed. BGL production was scaled up from a laboratory scale (7-L fermenter) to a pilot scale (70-L fermenter). When S. hirsutum was cultured in fed-batch culture with rice straw as the carbon source in a 70-L fermenter, a comparable productivity of 78.6 U/l/h was obtained. Furthermore, S. hirsutum showed high levels of activity of other lignocellulases (cellobiohydrolase, endoglucanase, xylanase, and laccase) that are involved in the saccharification of biomasses. Application of S. hirsutum lignocellulases in the hydrolysis of Pinus densiflora and Catalpa ovata showed saccharification yields of 49.7% and 43.0%, respectively, which were higher than the yield obtained using commercial enzymes.

Extracellular β-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology

beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.

Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli

A putative beta-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-beta-D-1-thiogalactopyranoside, the enzyme expressed at approximately 40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A(600) 12 in auto-inducing M9NG medium, beta-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the beta-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45 degrees C and 8.0, respectively, under the assay conditions. K(m) and k(cat) against p-nitrophenyl-beta-D-glucopyranoside were 4 mM and 0.75 sec(-1), respectively. To our knowledge, this is the first report of high-level expression and characterization of a beta-glucosidase from B. halodurans.

Influence of iron and copper nanoparticle powder on the production of lignocellulose degrading enzymes in the fungus Trametes versicolor

White rot fungi are one of the key group of microorganisms that help to enrich the soil via degradation of wood. In the current communication, influence of iron and copper nanoparticles on the production of lignocellulolytic enzymes by Trametes versicolor have been investigated. The production of enzymes in the presence of the two nanoparticles was compared to that of ferrous and cupric ions respectively. Results show that both the tested nanoparticles alter the production profile of the lignocellulolytic enzymes when compared to the control set. The production of laccase was not influenced by iron nanoparticles but was effected by copper nanoparticles within 24h of incubation. Both the nanoparticles decreased the production of beta-glucosidase, beta-xylosidase and cellobiohydrolase significantly. However, the production profile of Mn-peroxidase and remained statistically similar to that of control when the organism was incubated with iron and copper nanoparticles. The production profiles were also different when one compares the ionic form of metals and the nanoparticles, suggesting different mechanism of action of the particles on the organism. The difference in the production profile was not growth related as no significant difference was recorded for either form of iron and copper on the growth of T. versicolor.

Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Characterization of β-glucosidase from a strain of Penicillium purpurogenum KJS506

A novel beta-glucosidase (BGL)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal BGL activity of 12.3 U ml(-1), one of the highest levels among BGL-producing microorganisms was observed. The optimum temperature and pH for BGL production were 32 degrees C and 4, respectively. An extracellular BGL was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants, and the purified BGL showed higher activity (V (max) = 934 U mg protein(-1)) than most BGLs from other sources. The complete ORF of bgl3 was cloned from P. purpurogenum by a modified thermal asymmetric interlaced polymerase chain reaction. The bgl3 gene consists of a 2,571-bp ORF and encodes a putative protein containing 856 amino acids with a calculated molecular mass of 89,624 Da. The putative gene product was identified as a member of glycoside hydrolase family 3. The present results should contribute to improved industrial production of BGL by P. purpurogenum KJS506.

Production and distribution of β-glucosidase in a mutant strain Trichoderma viride T 100-14

The characterization of beta-glucosidase's production and distribution in a mutant strain Trichoderma viride T 100-14 at extracellular and intracellular levels were studied in this paper. Three experiment groups were done automatically with pH controlled at 4.8 during fermentation process, with 1mg/ml 2-deoxy-d-glucose addition or without pH control and 2-deoxy-d-glucose addition (control). Activity assay and electron microscopic immunogold labeling experiments were performed at different culture periods (24, 48, 72, 96 and 120 hours). Under constant pH 4.8, high density of immunogold labeling particles, highest intracellular enzyme activity, total enzyme activity and specific activity were observed at 24 hours of fermentation. After 72 hours, the extracellular and total activities fluctuated little and the maximal activity in extracellular fraction was 2.7 times higher than control. By contrast, with 2-deoxy-d-glucose addition, the secreted and total beta-glucosidase activities achieved their maximum at 96 hours of fermentation, and the maximal secreted activity increased 2.05-fold than the control. Additionally, the secretion ratio (maximal secreted beta-glucosidase activity/maximal total activity) with pH control or 2-deoxy-d-glucose addition was elevated profoundly near to a level as the cellulase in fungi.

Thiamine increases β-glucosidase production in the newly isolated strain ofFomitopsis pinicola

To isolate a high beta-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain. A high BGL-producing strain was isolated and identified as Fomitopsis pinicola KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, F. pinicola was supplemented with various vitamins. Supplementation with thiamine (20 mg l(-1)) improved BGL production in F. pinicola cultures by 3.7-fold to give a specific activity of 114.4 micromol min(-1) mg(-1) protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from F. pinicola culture showed the highest catalytic efficiency ever reported. Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, F. pinicola KMJ812. Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.