Addition Of Cellobiose Research Articles

Effect of different freezing conditions on ice crystal formation behavior and ice-growth inhibition by cryoprotectants.

The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long-term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low-temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze-thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice-growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice-growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry.

Enzymatic hydrolysis of microcrystalline cellulose for efficient and economic production of astragalin in metabolic engineering of Escherichia coli

Cellulose is the most abundant biomass in nature, and the development of high-value utilization technologies for cellulose is of great significance. In this work, a recombinant strain was engineered by introducing Arabidopsis thaliana glycosyltransferase (AtUGT78D2) and the cellobiose phosphorolysis route to produce astragalin from kaempferol. By optimizing the transformation conditions, the production of astragalin increased from 1053 to 3031 mg/L with the addition of cellobiose. Subsequently, the cellulose enzymatic solution was used to replace cellobiose as the carbon source and UDP-glucose precursor for producing astragalin. By optimizing enzymatic conditions and adding additives, the concentration of cellobiose significantly increased, resulting in an increase in the production of astragalin to 2279 mg/L. The adsorption strategy was employed to modulate the composition and activity of cellulase, and the ratio of cellobiose to glucose in the cellulose enzymatic solution increased from 0.73 to 1.62, with the yield of cellobiose reaching up to 5.9 g/L. Finally, the highest production of astragalin in the recombinant strain reached 2654 mg/L when the optimal ratio of cellobiose to glucose was used as the carbon source. This study provides a novel method for producing astragalin by using the enzymatic hydrolysis of microcrystalline cellulose.

MEDIUM OPTIMIZATION FOR ISOFLAVONE AGLYCONES PRODUCTION FROM FERMENTED DEFATTED SOYBEAN FLOUR BY DISPLAY OF Β-GLUCOSIDASE FROM BACILLUS LICHENIFORMIS NRC24 AND EVALUATION OF THEIR ANTIVIRAL AND ANTITUMOR ACTIVITIES

The submerged fermentation parameters of defatted soybean flour with Bacillus licheniformis NRC24 were optimized using the one-factor at-a-time method and Box-Behnken design to maximize the production of β-glucosidase and transformation of glucosidic isoflavones (daidzin and genistin) into their aglycones. One-factor-at-a-time optimization studies revealed that 80 g/L of defatted soybean flour , fermentation time of 16 h, temperature at 37°C, initial pH value of 7, inoculum volume of 5% (6x107 CFU/mL), and medium volume of 50 mL in 250 mL Erlenmeyer flask with shaking conditions at 100 rpm were the most suitable conditions for aglycones production. The addition of cellobiose (1 percent w/v) to the soy flour medium resulted in a significantly increase in total aglycone. Further optimization using the Box-Behnken design resulted in a 1.11-fold increase in daidzein and genistein. Antiviral activity was observed against the (H5N1) influenza, herpes simplex type 1, and rota virus. The MTT cytotoxicity assay of the extract using MDCK, Vero, and MA104 cell lines revealed that fermented soybean flour reduced cytotoxicity more than the unfermented one. However, it showed higher cytotoxicity against HCT-116 colon cancer cells, MCF-7 breast and PC3 prostate cancer cells than the unfermented soy extract.

The quality properties of frozen large yellow croaker fillets during temperature fluctuation cycles: improvement by cellobiose and carboxylated cellulose nanofibers

Frozen aquatic products undergo unavoidable quality changes owing to temperature fluctuations during frozen storage and distribution. This study investigated the effects of 1% cellobiose (CB), and 0.5 and 1% carboxylated cellulose nanofibers (CNF) on ice crystal growth and recrystallization of frozen large yellow croaker fillets exposed to temperature fluctuations. Denser and more uniformly distributed ice crystals were observed in the CB- and CNF-treated samples than in the water-treated samples. Furthermore, the addition of CB and CNF suppressed the conversion of bound water to frozen water in the samples during temperature fluctuation cycles, played a positive role in fixing the ionic and hydrogen bonds that stabilize the protein structure, limited the conformational transition from α-helix to β-sheet, and improved protein thermal stability. Based on turbidity, zeta potential, and confocal laser scanning microscopy (CLSM) analyses, the presence of CB and CNF restricted the protein aggregation. Compared with CB, CNF molecules with abundant carboxyl functional groups and longer morphology exhibited better cryoprotective effects. Moreover, the fillets were more improved protected from mechanical damage induced by large ice crystals at a higher CNF concentration. This study reveals the potential of CB and CNF as novel cryoprotectants.

Effect of cellobiose on the myofibrillar protein denaturation induced by pH changes during freeze-thaw cycles

The aim of this study was to investigate myofibrillar protein (MFP) denaturation induced by pH changes during freeze–thaw (FT) cycles, and to propose an effective mitigation strategy. Owing to the selective crystallization of Na2HPO4·12H2O and the consequent pH change, a pH change of 3.32 units was observed when the MFP solution were frozen. The surface hydrophobicity, particle size and confocal laser scanning microscopy showed that the protein molecules gradually unfolded and formed larger protein aggregation as the number of FT cycles increases. Additionally, protein degradation, secondary and tertiary structure alterations suggested that the FT cycle could disrupt structural integrity. The addition of cellobiose could maximize the inhibition of pH changes (decrease of ∼0.62 unit), no Na2HPO4·12H2O crystallization was observed by X-ray diffraction. Cellobiose could minimize FT damage to myofibrillar protein, which was closest to the control. Thus, cellobiose can be used as a new and effective cryoprotectant.

Dynamics of soil respiration and microbial communities: Interactive controls of temperature and substrate quality

Soil microbial communities mediate soil feedbacks to climate; a thorough understanding of their response to increasing temperatures is therefore central to predict climate-induced changes in carbon (C) fluxes. However, it is unclear how microbial communities will change in structure and function in response to temperature change and to the availability of organic C which varies in complexity. Here we present results from a laboratory incubation study in which soil microbial communities were exposed to different temperatures and organic C complexity. Soil samples were collected from two land-use types differing in climatic and edaphic conditions and located in two regions in southwest Germany. Soils amended with cellobiose (CB), xylan, or coniferyl alcohol (CA, lignin precursor) were incubated at 5, 15 or 25 °C. We found that temperature predominantly controlled microbial respiration rates. Increasing temperature stimulated cumulative respiration rates but decreased total microbial biomass (total phospholipid fatty acids, PLFAs) in all substrate amendments. Temperature increase affected fungal biomass more adversely than bacterial biomass and the temperature response of fungal biomass (fungal PLFAs, ergosterol and ITS fragment) depended upon substrate quality. With the addition of CB, temperature response of fungal biomass did not differ from un-amended control soils, whereas addition of xylan and CA shifted the fungal temperature optima from 5 °C to 15 °C. These results provide first evidence that fungi which decompose complex C substrates (CA and xylan) may have different life strategies and temperature optima than fungal communities which decompose labile C substrate (CB). Gram-positive and gram-negative bacteria differed strongly in their capacity to decompose CB under different temperature regimes: gram-positive bacteria had highest PLFA abundance at 5 °C, while gram-negative bacteria were most abundant at 25 °C. Bacterial community composition, as measured by 16S rRNA gene abundance, and PLFAs showed opposite temperature and substrate decomposition trends. Using multivariate statistics, we found a general association of microbial life strategies and key members of the microbial community: oligotrophic Alphaproteobacteria and Acidobacteria were associated with complex substrates and copiotrophic Actinobacteria with labile substrates. Our study provides evidence that the response of C cycling to warming will be mediated by shifts in the structure and function of soil microbial communities.

Characterization of a Lactococcus lactis promoter for heterologous protein production

Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Addition of cellobiose further increased the production of GFPmut3a by up to two-fold when compared to glucose. Analysis of mutations at two specific positions in the PTS-IIC promoter showed that a ‘T’ to ‘G’ mutation within the −35 element resulted in constitutive expression in glucose, while a ‘C’ at nucleotide 7 in the putative cre site enhanced promoter activity in cellobiose. Finally, this PTS-IIC promoter is capable of mediating protein expression in Bacillus subtilis and Escherichia coli Nissle 1917, suggesting the potential for future biotechnological applications of this element and its derivatives.

Novel Luciferase Reporter System for In Vitro and Organ-Specific Monitoring of Differential Gene Expression inListeria monocytogenes

In this paper we describe construction of a luciferase-based vector, pPL2lux, and use of this vector to study gene expression in Listeria monocytogenes. pPL2lux is a derivative of the listerial integration vector pPL2 and harbors a synthetic luxABCDE operon encoding a fatty acid reductase complex (LuxCDE) involved in synthesis of the fatty aldehyde substrate for the bioluminescence reaction catalyzed by the LuxAB luciferase. We constructed pPL2lux derivatives in which the secA and hlyA promoters were translationally fused to luxABCDE and integrated as a single copy into the chromosome of L. monocytogenes EGD-e. Growth experiments revealed that hlyA was expressed predominantly in the stationary phase in LB medium buffered at pH 7.4, whereas secA expression could be detected in the exponential growth phase. Moreover, the correlation between luciferase activity and transcription levels, as determined by reverse transcriptase PCR, was confirmed using conditions known to lead to repression and activation of hemolysin expression (addition of cellobiose and activated charcoal, respectively). Furthermore, hemolysin expression could be monitored in real time during invasion of an intact monolayer of C2Bbe1 (Caco-2-derived) cells. Finally, hemolysin expression could be detected in the livers, spleens, and kidneys of mice 3 days postinfection. These experiments clearly established the effectiveness of pPL2lux as a quantitative reporter system for real-time, noninvasive evaluation of gene expression in L. monocytogenes.

Enhancement of the viscometric endocellulase activity of Polyporus arcularius CMCase IIIa by cellobiose and cellooligosaccharides

Purification and viscometric characterization of three CMCases from Polyporus arcularius were carried out. The three CMCases, I, II, and IIIa, were estimated to have molecular masses of 39.1 kDa, 36.3kDa, and 24.3kDa, respectively. The addition of cellobiose and cellooligosaccharides to the reaction mixtures of CMCase I and II inhibited viscometric endocellulase activity. Following the addition of 20 mM cellobiose, CMCase I and II activities fell to about 30%–36% of their activity in the absence of cellobiose. CMCase IIIa activity, on the other hand, increased in proportion to the increase in cellobiose or cellooligosaccharide concentration. Maximal enhancement of CMCase IIIa activity was observed following the addition of cellobiose, whereas less enhancement was observed with cellooligosaccharides spanning more than two glucoside units. The addition of 20mM cellobiose resulted in an increase greater than 500% in CMCase IIIa activity. Inhibition of CMCase I and II by cellobiose and cellooligosaccharides may be the result of competition between the substrate and the reaction products. One of the reaction products of CMCase IIIa may bind to a site other than the active site of the enzyme, thus enhancing CMCase IIIa activity.

Heterologous expression and characterization of the endocellulase encoding gene cel3A from the basidiomycete Polyporus arcularius

The genomic and cDNA clones encoding the carboxymethyl cellulase (CMCase) gene (cel3A) of Polyporus arcularius were sequenced and characterized. The coding region of cel3A, composed of 1329 bp, was found to encode a polypeptide of 243 amino acids that has similarity with FI-CMCase of Aspergillus aculeatus. Expression of the cel3A cDNA in Escherichia coli led to production of a nonglycosylated protein as an active form; moreover, CMCase activity measured by the viscometric method was enhanced 4.47 times by addition of cellobiose. These results indicate that glycosylation and any modification of protein such as processing are not required for Cel3A activity.

A simple chemiluminescence-based method for rapid enumeration of Listeria spp. microcolonies.

Listeria monocytogenes is capable, under certain conditions, of producing chemiluminescence which is amplified by luminol. This property was used to detect and count microcolonies of Listeria spp. in a few hours, without the use of a microscope. After trapping Listeria cells on polyvinylidene fluoride membranes, a chemiluminescence mixture was sprayed onto the membrane. The chemiluminescent spots emitted were analysed by a charge-coupled device camera connected to a data-processing system, which restored the intensity of the signals into three dimensional images. The intensity of the luminescence of microcolonies was improved by addition of cellobiose, and by brief exposure to u.v. light. Microcolonies of Listeria spp. can be imaged and counted by luminol-enhanced chemiluminescence with a photon-counting system. This method can be applied to the rapid detection and counting of Listeria spp. in raw milk.

Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBH I (T.r. Cel 7A) in enantioseparations

Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of β-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.

Cellobiose dehydrogenase enhances Phanerochaete chrysosporium cellobiohydrolase I activity by relieving product inhibition.

The interaction of cellobiose dehydrogenase (CDH) with cellobiohydrolase I (CBH I) in cellulose-grown cultures of Phanerochaete chrysosporium was investigated to clarify the role of CDH in cellulose degradation. Decomposition of bacterial microcrystalline cellulose by CBH I was enhanced significantly in the presence of the CDH/ferricyanide redox-system compared with CBH I alone. To explain this phenomenon, a model system, using p-nitrophenyl-beta-D-cellobioside as a substrate, was elaborated for measurement of CBH I activity with and without the CDH redox-system. The activity of CBH I for hydrolysis of p-nitrophenyl-beta-D-cellobioside was also enhanced in the presence of the redox system. It was found that Km for hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I was lower in the presence than in the absence of the CDH/ferricyanide redox-system, 142 microM and 384 microM, respectively, while no significant difference was observed between the k(cat) values. These results indicate that cellulase activity is enhanced by an increased affinity for p-nitrophenyl-beta-D-cellobioside, rather than by an increased hydrolysis rate. This shows that cellobiose, the hydrolysis product, acts as a competitive inhibitor of the interaction between CBH I and p-nitrophenyl-beta-D-cellobioside. This was confirmed by addition of cellobiose, which was found to competitively inhibit hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I in the absence of the CDH redox system, and the Ki value for cellobiose inhibition was estimated to be 65 microM. However, this inhibition did not occur if cellobiose was incubated with CDH before addition of CBH I. It was concluded from these results that the reason for the enhancement of CBH I activity in the presence of the CDH redox system was that it relieves competitive inhibition of cellobiose by its oxidation to cellobionolactone.

Induction of α‐amylase by maltooligosaccharides in Thermomonospora curvata

The action pattern of the α‐amylase produced by Thermomonospora curvata is unique. Maltooligosaccharides (maltose to maltopentaose) were tested individually for their ability to induce α‐amylase in this thermophilic actinomycete. Maltotetraose was the most inductive followed by maltotriose. Maltose was a good inducer of amylase production when used as sole carbon source, but had relatively little inductive capacity in the presence of either glucose or cellobiose. When cellobiose was added during exponential growth on maltose, maltose utilization and extracellular α‐amylase accumulation were transiently inhibited. With maltotriose as the initial carbon source, addition of cellobiose did not inhibit the utilization of the trisaccharide; however, cellobiose, whether added during exponential growth or stationary phase, resulted in the rapid degradation of amylase when maltotriose was depleted from the medium. This inactivation did not appear to be a growth phase‐induced phenomenon because stationary phase cells in the absence of cellobiose maintained their peak extracellular amylase level. This cellobiose‐mediated α‐amylase inactivation would be particularly important during production of the enzyme on a complex lignocellulosic substrate.

Factors Affecting Microbial 2,4,6-Trinitrotoluene Mineralization in Contaminated Soil

The influence of selected environmental factors on microbial TNT mineralization in soils collected from a TNT-contaminated site at Weldon Spring, MO, was examined using uniformly ring-labeled [ 14 C]TNT. Microbial TNT mineralization was significantly inhibited by the addition of cellobiose and syringate. This response suggests that the indigenous microorganisms are capable of metabolizing TNT but preferentially utilize less recalcitrant substrates when available. The observed inhibition of TNT mineralization by TNT concentrations higher than 100 μmol/kg of soil and by dry soil conditions suggests that toxic inhibition of microbial activity at high TNT concentrations and the periodic drying of these soils have contributed to the long-term persistence of TNT at Weldon Spring. In comparisonto aerobic microcosms, mineralization was inhibited in anaerobic microcosms and in microcosms with a headspace of air amended with oxygen, suggesting that a mosaic of aerobic and anaerobic conditions may optimize TNT degradation at this site.

Purification and Characterization of Cellobiose Dehydrogenase, a Novel Extracellular Hemoflavoenzyme from the White-Rot Fungus Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH), an extracellular hemoflavoenzyme produced by the cellulose-degrading cultures of Phanerochaete chrysosporium, oxidizes cellobiose to cellobionolactone. CDH has been purified to homogeneity by a five-step purification procedure. The homogeneous CDH is monomeric and has a relative molecular mass of 90,000. It is also a glycoprotein with a neutral carbohydrate content of 9.4%. Purified CDH contains one heme b and one flavin adenine dinucleotide per monomer. Homogeneous CDH has a specific activity of 10.3 μmol min−1 mg−1 for cytochrome c reduction, in the presence of cellobiose. Cellotriose, cellotetraose, cellopentaose, and lactose also serve as substrates for CDH, in addition to cellobiose. Cytochrome c, dichlorophenolindophenol, Mn3+, and benzoquinones can function as electron acceptors in these oxidations. Kinetic studies suggest that cellobiose is the preferred substrate and cytochrome c is the preferred electron acceptor. In the absence of these electron acceptors, oxygen serves as a poor electron acceptor and is reduced to H2O2. CDH is very stable in the pH range 3-10 and up to 50°C. At lower pH or at higher temperature, CDH is inactivated due to the release of flavin from the active site. The native ferric form of the enzyme has absorption maxima at 420, 529, and 570 nm. With the addition of cellobiose, these absorptions shift to 428, 534, and 564 nm. The ferric enzyme does not bind azide or cyanide, implying that the heme iron is probably hexacoordinate.

Origin of oxidized cellulose degradation products and mechanism of their promotion of cellobiohydrolase I biosynthesis in Trichoderma reesei

Cellobiono-1,5-lactone (CBL) induces cellulase, particularly cellobiohydrolase I (CBH I) formation in Trichoderma reesei. When used as a sole source of carbon, it promoted comparably poor growth, because (a) the δ-gluconolactone arising by the action of β-glucosidase is not metabolized by the fungus, and (b) it is a low-V max substrate of the T. reesei β-glucosidase. Induction of higher amounts of CBH I were observed when CBL was supplied as carbon source together with cellobiose than when supplied alone. Maximal CBH I levels formed under these conditions were comparable to those when T. reesei was grown on cellobiose plus β-gluconolactone, an inhibitor of β-glucosidase. These data suggest an indirect effect of CBL on CBH I induction, probably by inhibiting cellobiose hydrolysis. The origin of CBL during growth of T. reesei on cellulose was also investigated: aldonic acids and aldonolactones were released from cellulose by T. reesei culture fluids. Artificially oxidized celluloses gave rise to the appearance of higher concentrations of oxidized products but the addition of cellobiose did not, suggesting that their appearance is not due to a cellobiose-oxidizing enzyme. No accumulation of oxidized cello-oligosaccharides occurred when the action of both cellobiohydrolases (CBH I and II) was simultaneously blocked by monoclonal antibodies. These data suggest that cellulose already contains oxidized chain termini, and that these aldonolactones and aldonic acids are released by the action of the two cellobiohydrolases and not by a ‘cellulose-oxidizing’ enzyme.

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3- O-methylglucoside. Cellodextrins (C 2 to C 6) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [ 3H]cellobiose, [ 3H]sophorose, and [ 14C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3- O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [ 3H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.

Effect of peat extract on the hydrolytic enzymes of Phanerochaete chrysosporium

Abstract Peat, a partially decomposed plant material rich in minerals and lignocellulose was tested as a substrate for the growth and production of hydrolytic enzymes viz. cellulase, cellobiase and xylanase in Phanerochaete chrysosporium. Three types of peak extracts such as cold, hot water and autoclaved were prepared and tested. Among them, autoclaved extract supported the maximal growth. The intracellular enzyme activities peaked on the fifth day after inoculation irrespective of the media and enzyme tested. Addition of cellobiose and lactose in the medium induced the production of cellulase, xylanase and to a lesser extent cellobiase. Addition of glucose and sucrose to the media resulted in the suppression of all the extracellular enzymes tested.

A thermostable ?-glucosidase isolated from a bacterial species of the genus Thermus

An extremely thermophilic aerobic bacterium which produced β-glucosidase was isolated from soil collected at the Yudanaka hot spring in Japan. It was identified as belonging to the genus Thermus. Production of β-glucosidase by this bacterium was stimulated by the addition of cellobiose or laminaribiose to the medium. The optimum pH and temperature of the enzyme were 4.5–6.5 and 85° C respectively. The enzyme was stable in the pH range of 4.5–7.0 at 70° C for 2 h and the half-life at 75° C was 5 days. The Km value of the enzyme for p-nitrophenyl-β-d-glucopyranoside, determined at 70° C in 0.1 M sodium phosphate buffer (pH 6.5), was 0.28 mM while the Km was 2.0 mM for cellobiose. The enzyme effectively hydrolysed cellobiose at 70° C and the conversion yields of cellobiose to glucose were 95%, 93% and 90% at substrate concentrations of 5%, 10% and 15%, respectively.