Processive Endoglucanases Mediate Degradation of Cellulose by Saccharophagus degradans

Bacteria and fungi are thought to degrade cellulose through the activity of either a complexed or a noncomplexed cellulolytic system composed of endoglucanases and cellobiohydrolases. The marine bacterium Saccharophagus degradans 2-40 produces a multicomponent cellulolytic system that is unusual in its abundance of GH5-containing endoglucanases. Secreted enzymes of this bacterium release high levels of cellobiose from cellulosic materials. Through cloning and purification, the predicted biochemical activities of the one annotated cellobiohydrolase Cel6A and the GH5-containing endoglucanases were evaluated. Cel6A was shown to be a classic endoglucanase, but Cel5H showed significantly higher activity on several types of cellulose, was the highest expressed, and processively released cellobiose from cellulosic substrates. Cel5G, Cel5H, and Cel5J were found to be members of a separate phylogenetic clade and were all shown to be processive. The processive endoglucanases are functionally equivalent to the endoglucanases and cellobiohydrolases required for other cellulolytic systems, thus providing a cellobiohydrolase-independent mechanism for this bacterium to convert cellulose to glucose.The microbial degradation of cellulose is of interest due to applications in the sugar-dependent production of alternative biofuels (25). There are well-characterized cellulolytic systems of bacteria and fungi that employ multiple endo-acting glucanases and exo-acting cellobiohydrolases in the degradation of cellulose (12). For example, the noncomplexed cellulase system of the wood soft rot fungus Hypocrea jecorina (anamorph Trichoderma reesei), the source for most commercially available cellulase preparations, produces up to eight secreted β-1,4-endoglucanases (Cel5A, Cel5B, Cel7B, Cel12A, Cel45A, Cel61A, Cel61B, and Cel61C), two cellobiohydrolases (Cel6A and Cel7A), and several β-glucosidases (e.g., Bgl3A) (21). Cellobiohydrolases are critical to the function of these systems, as, for example, Cel7A comprises in excess of 50% of the cellulases secreted by this organism (11). Another well-characterized noncomplexed cellulase system is found in Thermobifida fusca, a filamentous soil bacterium that is a major degrader of organic material found in compost piles (32). This bacterium also secretes several endoglucanases and end-specific cellobiohydrolases to degrade cellulose (32). An alternative mechanism for degradation of cellulose is found in microorganisms producing complexed cellulolytic systems, such as those found in cellulolytic clostridia. In these microorganisms, several β-1,4-endoglucanases and cellobiohydrolases assemble on surface-associated scaffoldin polypeptides to form cellulose-degrading multiprotein complexes known as cellulosomes (2, 6). The unifying theme in both complexed and noncomplexed systems is the importance of cellobiohydrolases in converting cellulose and cellodextrins to soluble cellobiose.Recently, a complete cellulolytic system was reported to occur in the marine bacterium Saccharophagus degradans 2-40 (28, 31). This bacterium is capable of growth on both crystalline and noncrystalline celluloses as sole carbon sources and produces multiple glucanases that can be detected in zymograms of cell lysates (28). The genome sequence of this bacterium predicts that the cellulolytic system of this bacterium consists of 10 GH5-containing β-1,4-endoglucanases (Cel5A, Cel5B, Cel5C, Cel5D, Cel5E, Cel5F, Cel5G, Cel5H, Cel5I, and Cel5J), two GH9 β-1,4-endoglucanases (Cel9A and Cel9B), one cellobiohydrolase (Cel6A), five β-glucosidases (Bgl1A, Bgl1B, Bgl3C, Ced3A, and Ced3B), and a cellobiose phosphorylase (Cep94A) (28, 31). The apparent absence of a homolog to a scaffoldin in the genome sequence and to dockerin-like domains in the proposed glucanases suggests that this bacterium produces a noncomplexed cellulolytic system. Two unusual features of this cellulolytic system are the large number of GH5 endoglucanases and the presence of only one annotated cellobiohydrolase, Cel6A (28, 31). The apparent deficiency of cellobiohydrolases in this system raised the question as to the mechanism by which this bacterium degrades cellulose.To understand the mechanism for degradation of cellulose, the biochemical activities for the predicted cellobiohydrolase Cel6A and each of the GH5 glucanases predicted for the S. degradans cellulolytic system were evaluated. Cel6A exhibited properties of a classic endoglucanase, but three of the originally annotated endoglucanases, Cel5G, Cel5H, and Cel5J, were shown to be processive, forming cellobiose as the end product. Processive endoglucanases substitute for cellobiohydrolases in this system to play a major role in the degradation of cellulose.     

Carbon source directs the differential expression of β-glucosidases in <Emphasis Type="Italic">Stachybotrys microspora</Emphasis>

Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple β-glucosidases. The production of these enzymes was studied under culture with variable carbon sources. The highest activity was produced on glucose (0.66 U ml⁻¹) whereas galactose, lactose, cellobiose, Avicel cellulose, carboxymethylcellulose (CMC), wheat bran and gruel allowed intermediate production levels ranging between 0.08 and 0.48 U ml⁻¹. The zymogram analysis showed that complex sugars such as Avicel cellulose and CMC induced the expression of several β-glucosidases whereas all tested simple sugars (mono and disaccharides) induced the expression of one single β-glucosidase, each time different. The most efficient β-glucosidase named bglG was produced on glucose which continues to be, at the same time, its strong inhibitor. The bglG N-terminal sequence confirmed that it is a novel β-glucosidase. According to its large molecular weight, this enzyme was assumed to belong to family 3 of β-glucosidases. RT-PCR analysis showed that family 3 expressions were induced on glucose while those of family 1 were repressed. Finally, with the use of different combinations of glucose and various carbon sources at different ratio, we showed that such sources direct the differential expression of β-glucosidases in S. microspora since our strain co-produced the β-glucosidases corresponding to each carbon source.     

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0–5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.     

Purification and biochemical characterization of a transglucosilating β-glucosidase of <Emphasis Type="Italic">Stachybotrys</Emphasis> strain

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K _m and V _max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min⁻¹ ml⁻¹. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.     

A novel thermostable and glucose-tolerant β-glucosidase from <Emphasis Type="Italic">Fervidobacterium islandicum</Emphasis>

An open reading frame (ORF) encoding the enzyme β-glucosidase from the extremely thermophilic bacterium Fervidobacterium islandicum has been identified, cloned and sequenced. The bgl1A gene was cloned in a pET-Blue1 vector and transformed in Escherichia coli, resulting in high-level expression of β-glucosidase FiBgl1A that was purified to homogeneity in a two-step purification. FiBgl1A is composed of 459 amino acid residues and showed high homology to glycoside hydrolase family 1 proteins. It exhibited highest activity towards p-nitrophenyl-β-d-glucopyranoside with an optimum activity at pH 6.0 and 7.0 and at 90 °C. The enzyme is resistant to glucose inhibition. Furthermore, it did not require divalent cations for activity, nor was it affected by the addition of p-chloromercuribenzoate (10 mM), EDTA (10 mM), urea (10 mM) or dithiothreitol (10 mM). Addition of surfactants (with the exception of SDS) and a number of solvents enhanced the activity of FiBgl1A. It also displayed remarkable activity across a broad temperature range (80–100 °C). The thermoactivity and thermostability of FiBgl1A and its resistance to denaturing and reducing agents make this enzyme a potential candidate for industrial applications.     

Identification and functional analysis of a gene encoding β-glucosidase from the brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.     

Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi

Thirteen thermophilic and thermotolerant fungal cultures isolated from composting soils produced diverse β-glucosidases as indicated by zymograms of PAGE developed using 4-methylumbelliferyl-β-d-glucoside. IEF profiling revealed the presence of 28 β-glucosidases separated on the basis of their pI. Eleven of the β-glucosidases were active under acidic conditions. Two β-glucosidase isoforms, ASCβG-II of Aspergillus caespitosus and HIβG-I of Humicola insolens were resistant to inhibition by glucose and were active in the presence of 300 and 100 (mM) glucose, respectively.     

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase.     

Hydrolysis of cellulose in synergistic mixtures of β-glucosidase and endo/exocellulase Cel9A from <Emphasis Type="Italic">Thermobifida fusca</Emphasis>

The synergism between the endo/exocellulase, Cel9A, and β-glucosidase (βgl) of Thermobifida fusca was investigated. Wild type βgl or S319C, a βgl mutant with significantly improved cellobiase activity, were added to Cel9A. Both wild type and mutant βgl enhanced the Cel9A hydrolysis of carboxymethyl cellulose (CMC) and filter paper by 50–100% compared to Cel9A alone. No enhancement occurred with addition of E388A, an inactive form of βgl. HPLC analysis showed that, with Cel9A alone, the resulting hydrolysate of glucose and cellobiose contained about half glucose; after addition of equimolar amounts of either wild type βgl or mutant S319C to Cel9A, the hydrolysate contained more than 85% glucose. βgl thus acted synergistically with Cel9A by converting cello-oligomers to glucose; this reduced the soluble sugar accumulation during hydrolysis of cellulose.     

Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana

The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.     

Immobilization of β-glucosidase on Eupergit C for Lignocellulose Hydrolysis

β-Glucosidase is frequently used to supplement cellulase preparations for hydrolysis of cellulosic and lignocellulosic substrates in order to accelerate the conversion of cellobiose to glucose. Typically, commercial cellulase preparations are deficient in this enzyme and accumulation of cellobiose leads to product inhibition. This study evaluates the potential for recycling β-glucosidase by immobilization on a methacrylamide polymer carrier, Eupergit C. The immobilized β-glucosidase had improved stability at 65 °C, relative to the free enzyme, while the profile of activity versus pH was unchanged. Immobilization resulted in an increase in the apparent K_m from 1.1 to 11 mm and an increase in V_max from 296 to 2430 μmol mg⁻¹ min⁻¹. The effect of immobilized β-glucosidase on the hydrolysis of cellulosic and lignocellulosic substrates was comparable to that of the free enzyme when used at the same level of protein. Operational stability of the immobilized β-glucosidase was demonstrated during six rounds of lignocellulose hydrolysis. Received 22 August 2005; Revisions requested 20 September 2005; Revisions received 8 November 2005; Accepted 10 November 2005     

Cloning and expression of β-glucosidase genes inEscherichia coli andSaccharomyces cerevisiae using shuttle vector pYES 2.0

Genes for β-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium,Cellulomonas biazotea, were cloned in pUC18 in itsSacI cloning site and transformed toE. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representativeE. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of β-glucosidase onE. coli. Expression of thebgl genes resulted in overproduction of β-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carryingbgl genes from the representative recombinantE. coli were isolated withSacI ligated in the shuttle vector pYES2.0 in itsSacI site and transformed toE. coli andS. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of β-glucosidase onS. cerevisiae and enabled it to grow on cellobiose and salicin. Thegall promoter of shuttle vector pYES2.0 enabled the organisms to produce twice more β-glucosidase than that supported by thelacZ-promoter of pUC18 plasmid inE. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains ofS. cerevisiae The enzyme produced bybgl ⁺ yeast andE. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the β-glucosidases fromS. cerevisiae were substantially the same as those fromC. biazotea.     

Production of Cellulases by Aspergillus fumigatus and Characterization of One β-Glucosidase

Aspergillus fumigatus produces substantial extracellular cellulases on several cellulosic substrates including simple sugars. Low glucose potentiates enzyme production, but most cellulose-induced cellulases are repressed by high glucose. As production of cellulase in a wide substrate range is unusual, the cellulolytic complex of this thermophilic fungus was investigated. A β-glucosidase was separated by gel filtration and ion-exchange chromatography. It migrated in native polyacrylamide gel as a single protein (130 kDa), which split under denaturing conditions into two smaller proteins having molecular masses of 90 kDa and 45 kDa. However, only the 90-kDa protein was active. Conventional chromatographic procedures were unsuccessful for the separation of these two proteins. Therefore, the 130-kDa protein was studied for its kinetic properties. It hydrolyzed p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose, but not β-glucans, laminarin, and p-nitrophenyl-β-D-xilopyranoside. The optimal pH and temperature of p-NPG and cellobiose hydrolysis were 5.0 and 4.0, and 65°C and 60°C, respectively. The K _m values, determined for cellobiose and p-NPG of hydrolysis, were 0.075 mM and 1.36 mM, respectively. Glucose competitively inhibited the hydrolysis of p-NPG. The K_i was 3.5 mM.     

Efficient synthesis of gluco-oligosaccharides and alkyl-glucosides by transglycosylation activity of β-glucosidase from <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

An extracellular β-glucosidase (β-glu x) from Sclerotinia sclerotiorum was used as catalyst for the synthesis of gluco-oligosaccharides (GOSs) and alkyl-glucosides. The purified β-glu x was not regiospecific for β(1→4) linkages in either hydrolysis or transglycosylation catalysed-reactions. It efficiently synthesized GOSs from cellobiose, gentiobiose and methyl β-d-glucoside by transglycosylation. At optimal conditions, 119 mg/ml of GOSs (∼ ∼33%) were formed over 9 h from cellobiose as substrate. Alkyl-glucosides were also efficiently synthesized by transglycosylation of cellobiose in presence of different alcohols in biphasic media. However, their concentrations decreased as the size of the alcohol chain increased.     

Expression and characterization of a glucose-tolerant β-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and β-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a β-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most β-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K _m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10⁻², 5.6 × 10⁻², and 5.3 × 10⁻² mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and β-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.

     

Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillium brasilianum

A GH3 β-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60°C at pH 4–6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-β-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-¹³C₆ as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.     

Degradation of rice bran hemicellulose by <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. strain HC1: gene cloning,characterization and function of β-D-glucosidase as an enzyme involved in degradation

A bacterium (strain HC1) capable of assimilating rice bran hemicellulose was isolated from a soil and identified as belonging to the genus Paenibacillus through taxonomical and 16S rDNA sequence analysis. Strain HC1 cells grown on rice bran hemicellulose as a sole carbon source inducibly produced extracellular xylanase and intracellular glycosidases such as β-d-glucosidase and β-d-arabinosidase. One of them, β-d-glucosidase was further analyzed. A genomic DNA library of the bacterium was constructed in Escherichia coli and gene coding for β-d-glucosidase was cloned by screening for β-d-glucoside-degrading phenotype in E. coli cells. Nucleotide sequence determination indicated that the gene for the enzyme contained an open reading frame consisting of 1,347 bp coding for a polypeptide with a molecular mass of 51.4 kDa. The polypeptide exhibits significant homology with other bacterial β-d-glucosidases and belongs to glycoside hydrolase family 1. β-d-Glucosidase purified from E. coli cells was a monomeric enzyme with a molecular mass of 50 kDa most active at around pH 7.0 and 37°C. Strain HC1 glycosidases responsible for degradation of rice bran hemicellulose are expected to be useful for structurally determining and molecularly modifying rice bran hemicellulose and its derivatives.     

Two β-glucosidase activities in Fibrobacter succinogenes S85

Few bacteria are capable of degrading crystalline cellulose but there is considerable interest in the properties of enzyme systems with this capability. In the bovine and ovine rumen the principal cellulolytic bacterium is Fibrobacter (formerly Bacteroides ) succinogenes. The cellulase system of this organism is composed of multiple enzyme components, including a constitutive and cell-associated β -glucosidase active against cellobiose. The properties of the β -glucosidase activity have been investigated with the chromogenic substrate β -nitrophenyl β -D-glucoside (pNPG). Hydrolytic activity against pNPG was located primarily in the cytoplasm and the cytoplasmic membrane but showed a gradual migration to the periplasm during growth on either glucose or cellobiose. Activity against cellobiose was found in the periplasm in significant amounts in all growth phases. Of the β -glucosides tested, only cellobiose and pNPG were hydrolysed by crude cell extracts. In the presence of cellobiose, however, the rate of hydrolysis of pNPG was stimulated up to 10-fold, and extracts hydrolysed methylumbelliferyl β -D-glucoside, 5-bromo-4-chloro-3-indolyl β -D-glucoside, arbutin and aesculin. Activities against pNPG in the presence and absence of cellobiose displayed similar instability in the presence of oxygen; both were stabilized by dithiothreitol and the temperature and pH optima were identical. A significant proportion of the membrane-associated β -glucosidase was released by treatment with 0.3 mol/1 KCl, and fractionation by chromatography on CM-cellulose showed the presence of two activities against pNPG, only one of which was stimulated by cellobiose.     

Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg⁻¹ by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80oC, and the half-life at 70oC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l⁻¹ galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l⁻¹ glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l⁻¹), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l⁻¹) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.     

Characterization of β-glucosidases from Hanseniaspora sp. and Pichia anomala with potentially aroma-enhancing capabilities in juice and wine

The properties of intracellular β-glucosidases produced from two yeast isolates identified as Hanseniaspora sp. BC9 and Pichia anomala MDD24 were characterized. β-Glucosidase from Hanseniaspora sp. BC9 was not inhibited by both 20% w/v fructose and 20% w/v sucrose and was slightly inhibited by glucose (> 40% relative β-glucosidase activity with 10% w/v glucose). β-Glucosidase from P. anomala MDD24 was inhibited by glucose, fructose and sucrose. In the presence of 4–12% v/v ethanol, β-glucosidase from P. anomala MDD24 was stimulated in range 110–130% relative activity whereas β-glucosidase from Hanseniaspora sp. BC9 was substantially inhibited in the presence of ethanol. Finally, juice and wine of the Muscat-type grape variety, Traminette, were selected to determine sugar-bound volatile aroma release, particularly terpenes, by the activity of those β-glucosidases. The results showed that high concentration of free aroma compounds were detected from Traminette juice treated with β-glucosidase from Hanseniaspora sp. BC9 and Traminette wine treated with β-glucosidase from P. anomala MDD24. The preliminary results with proposed an application of these enzymes in commercial wine production lead to more efficient of β-glucosidase from Hanseniaspora sp. BC9 in releasing desirable aromas during an early stage of alcoholic fermentation while β-glucosidase from P. anomala MDD24 is suitable at the final stage of alcoholic fermentation.