Structural changes of cellobiohydrolase I (1,4-β-<Emphasis Type="Italic">D</Emphasis>-glucan-cellobiohydrolase I,CBHI) and PNPC (<Emphasis Type="Italic">p</Emphasis>-nitrophenyl-β-<Emphasis Type="Italic">D</Emphasis>-cellobioside) during the binding process

Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demonstrated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.     

Structural changes of cellobiohydrolase I (1,4-beta-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitrophenyl-beta-D-cellobioside) during the binding process

Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demon-strated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.     

Exoglucanases fromZea mays L. seedlings: their role inβ-D-glucan hydrolysis and their potential role in extension growth

Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall β-D-glucan and their possible role in extension growth. An exo-β-1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both β1–4 and β1–3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-β-D-glucanase. Similarly, the capacity of the purified exo-β-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-β-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a β-glucosidase and did not appear to participate in the hydrolysis of wall β-D-glucan.     

Use of novel immobilized beta-galactosidase reactor to hydrolyze the lactose constituent of skim milk

beta-galactosidase from Aspergillus oryzae immobilized in an axial-annular flow reactor was used to effect the hydrolysis of the lactose component of skim milk. Nonlinear regression methods were employed to determine the kinetic parameters of four rate expressions derived from a proposed enzymatic mechanism. Data taken at three different temperatures (30 degrees C, 40 degrees C, and 50 degrees C) were fit via nonlinear regression methods assuming an Arrhenius temperature model for each of the parameters. For the reaction conditions used in this research, a three-parameter rate expression which includes the separate competitive inhibition effects of alpha- and beta-galactose (and the associated mutarotation reaction) is sufficient to model the hydrolysis of lactose in skim milk. The effects of temperature on the individual kinetic parameters are small. The most significant effect appears in the term for inhibition by the beta anomer of galactose (E(A) = 10.3 kcal/mol). At 40 degrees C and a space time of 10 min, 70% of the lactose present in skim milk can be hydrolyzed with the axial-annular flow reactor. This reactor can be used to hydrolyze the lactose in skim milk without the problems observed with other reactor configurations, namely, plugging due to particulates, microbial contamination, and large pressure drop.     

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and β-glucosidase played an important role in mannan hydrolysis. Addition of 10 mg/ml mannan reduced the glucose yield of Avicel (at 20 g/l) from 40.1 to 24.3%. No inhibition of β-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on β-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.     

Characterization and enzymatic hydrolysis of hydrothermally treated β-1,3–1,6-glucan from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

The chemical structure of hydrothermally treated β-1,3–1,6-glucan from Aureobasidium pullulans was characterized using techniques such as gas chromatography/mass spectrometry (GC/MS) and nuclear magnetic resonance (NMR). The chemical shifts of anomeric carbons observed in the ¹³C-NMR spectra suggested the presence of single flexible chains of polysaccharide in the sample. β-1,3–1,6-Glucan from A. pullulans became water-soluble, with an average molecular weight of 128,000 Da after hydrothermal treatment, and the solubility in water was approximately 10% (w/w). Sample (3% w/v) was completely hydrolyzed to glucose by enzymatic reaction with Lysing enzymes from Trichoderma harzianum. Gentiobiose (Glcβ1 → 6Glc) and glucose were released as products during the reaction, and the maximum yield of gentiobiose was approximately 70% (w/w). The molar ratio of gentiobiose to glucose after 1 h reaction suggested that the sample is likely highly branched. Sample (3% w/v) was also hydrolyzed to glucose by Uskizyme from Trichoderma sp., indicating that it is very sensitive to enzymatic hydrolysis.     

A comparison of characteristic temperatures for transport in two unsaturated fatty acid auxotrophs of escherichia coli

The rate of sugar transport as a function of temperature has been compared in two unsaturated fatty acid auxotrophs. One of these, the parent strain 30E, can β-oxidize the unsaturated fatty acid supplements, whereas the β-oxidation defective progeny strain 30Eβox^? cannot. In a previous study, Arrhenius plots for transport of β-glucosides and β-galactosides by strain 30Eβox^? revealed striking departures from linearity at both a lower and an upper characteristic temperatures. By electron spin resonance (esr) these temperatures were shown to correlate with the temperatures where the membrane lipids undergo a transition from a totally solid state to a solid–liquid equilibrium and from a solid–liquid equilibrium to a totally liquid state, respectively (1). In the present study with strain 30E we have made the following observations:

• 1 Arrhenius plots for transport rate are usually more complex, often revealing three characteristic temperatures. Two of these correlate with the upper and lower characteristic temperatures observed in strain 30Eβox^?. The third characteristic temperature falls between the previously described upper and lower ones.
• 2 In cells supplemented during growth with elaidate, the third characteristic temperature was identical within experimental limits for both β-glucoside and β-galactoside transport. indicating that it is likely to arise from some interaction in the bulk lipid phase. This conclusion is supported by the fact that the boundary of a change in physical state is also observed at this temperature by electron spin resonance.
• 3 In cells supplemented during growth with oleate, two or three characteristic temperatures were observed depending upon the transport system studied. Although glucoside and galactoside transport had the same lower characteristic temperature, these systems had no common upper characteristic temperature.
• 4 In cells supplemented during growth with the lipid density label, bromostearic acid, three characteristic temperatures were observed for β-glucoside transport in both strains 30E and 30Eβox^?.

     

Production of xylanolytic enzymes in association with the cellulolytic activities of Penicillium funiculosum

Penicillium funiculosum produced 16 and 0.4 units ml^?1 of d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37), respectively, in shake flasks. Both enzymes were 100% stable when heated at 50°C for 30 min and on prolonged heating d-xylanase and β-d-xylosidase showed 46 and 20% loss, respectively. Maximum hydrolysis (75%) of d-xylan was obtained when the end products were removed. The addition of β-d-xylosidase markedly influenced the degree of hydrolysis of d-xylan. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylose, d-xylobiose, d-xylotriose, d-xylotetraose, d-xylopentose and l-arabinose. The fractionation of culture filtrate of Penicillium funiculosum grown on cellulose powder or in a combination of cellulose powder and wheat bran indicated the presence of two d-xylanases. The role of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase on the overall hydrolysis of pure cellulose and lignocellulosic substrates is discussed.     

Efficient synthesis of octyl-β-d-galactopyranoside by Bacillus spore-displayed β-galactosidase using an amphiphilic 1,2-dimethoxyethane co-solvent

For enzymatic synthesis of octyl-β-d-galactopyranoside (octyl-gal) from lactose and n-octanol, Escherichia coli β-galactosidase (β-Gal) was expressed and displayed on the surfaces of Bacillus subtilis spores. The spore-displayed β-Gal was found to be stable when an amphiphilic 1,2-dimethoxyethane (DME) was used as a co-solvent; the transgalactosylation efficiency and octyl-gal conversion were optimal at 50% (v/v) DME. In addition, the product was maximally obtained from 100mM lactose in a phosphate buffer/n-octanol/DME (25/25/50, v/v) mixture. By increasing the agitation speed and the amount of spores displaying β-Gal, a yield of 33.7 mM octyl-gal was obtained over 24h in a batch mode, which is much higher than in other octyl-gal bioconversion processes, such as those involving lipid-coating, reverse micelles, or whole cells. On the other hand, intermittent addition of spore-displayed β-Gal and/or lactose in the reaction medium had no effect on the octyl-gal yield. The synthesized octyl-gal was hydrolyzed by the spore-displayed β-Gal, and a high concentration of octyl-gal competitively inhibited the enzymes (K(i) value of 10.8mM). In summary, we demonstrate that octyl-gal synthesis by spore-displayed β-Gal in non-aqueous medium can be significantly improved with the use of DME as a co-solvent.     

Fungal and other β-d-glucosidases — Their properties and applications

β-d-Glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) has been described in a variety of fungi and bacteria. Its function — to catalyse the hydrolysis of cellobiose, and aryl and alkyl β-d-glucosides — depends upon the nature of its source. Recent interest in this enzyme centres on its role in the enzymatic hydrolysis of cellulose. The rate and extent of cellulose hydrolysis can be increased by supplementing commercial cellulases with immobilized β-d-glucosidase, which has high stability and can be recovered and reused. The current state of β-d-glucosidase biotechnology is described.     

Enzymatic and molecular characterization of an endo-1,3-β-d-glucanase from the crystalline styles of the mussel Perna viridis

The retaining endo-1,3-β-d-glucanase (EC 3.2.1.39) was isolated from the crystalline styles of the commercially available Vietnamese edible mussel Perna viridis. It catalyzes hydrolysis of β-1,3-bonds in glucans and enables to catalyze a transglycosylation reaction. Resources of mass-spectrometry for analysis of enzymatic products were studied. cDNA sequence of endo-1,3-β-d-glucanase was determined by RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) methods. The cDNA of 1380bp contains an open reading frame of 1332bp encoding a mature protein of 328 amino acids. On basis of amino acid sequence analysis endo-1,3-β-d-glucanase was classified as a glycoside hydrolase of family 16.     

Simultaneous saccharification and fermentation of cellulose: effect of β-d-glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β-d-glucosidase to filter paper activity was ～1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ^?1, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

The Biochemistry of Parasites

HPLC-analysis of the reaction products of a series of 4-methylumbelliferyl glycosides from cello-oligosaccharides, used as substrates of a cellobiohydrolase from Trichoderma reesei, proves the lack of specificity for terminal cellobiosyl groups. Also, different reaction patterns are observed for this CBHI and for an endocellulase, when acting on these same substrates. 4-Methylumbelliferyl β-D-lactoside is an unexpected substrate for CBHI, yielding only lactose and phenol as reaction products. The binding characteristics of p-nitrobenzyl 1-thio-β-D-lactoside for this enzyme are determined by a dia-filtration technique, yielding 1 binding site and an association constant of 4.0 × 10⁴ M^?1.     

Does change in accessibility with conversion depend on both the substrate and pretreatment technology?

The accessibility of cellulase and xylanase enzymes to glucan and xylan, respectively, and its change with conversion were measured for pure Avicel glucan and poplar solids that had been pretreated by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute acid, and lime. Avicel and pretreated solids were digested to various degrees by cellulase together with β-glucosidase enzymes and then cleaned of residual protein via a biological method using Protease. Glucan accessibility was determined by purified CBHI (Cel7A) adsorption at 4 °C, and 4 and 24 h hydrolysis yields were determined for solids loading containing equal amounts of glucan (1.0% w/v) and lignin (1.0% w/v), in two separate sets of experiments. Consistent with our previous study and in contrast to some in the literature, little change in glucan accessibility was observed with conversion for Avicel, but glucan and xylan accessibility for real biomass varied with the type of pretreatment. For example, AFEX pretreated solids showed a negligible change in glucan accessibility for conversion up to 90%, although xylan accessibility seemed to decline first and then remained constant. On the other hand, a substantial decline in glucan and xylan accessibility with conversion was observed for lime pretreated poplar solids, as shown by initial hydrolysis rates. Yet, an increase in CBHI adsorption with conversion for lime pretreated poplar solids suggested the opposite trend, possibly due to increased lignin exposure and/or reduced effectiveness of adsorbed enzyme.     

Thermodynamic and kinetic aspects of the hydrolysis of ethyl(R)-2- (benzyloxycarbonylamino)-3-sulfamoylpropionate in the presence of free and immobilized alpha-chymotrypsin

The hydrolysis of ethyl (R)-2-(benzyloxycarbonylamino)-3-sulfamoylpropionate (blocked cysteic acid S-amide) by native and immobilized alpha-chymotrypsin was studied. The experiments were performed using a constant enzyme/substrate ratio of 1:8 and at a temperature of 10-40 degrees C; the immobilized enzyme was bound to a dialdehyde cellulose matrix. A kinetic equation (Eq.10) was found to be applicable which confirms that the mechanism of the enzyme reaction consists of several stages, irrespective of the enzyme state. The temperature dependence of the reaction velocity was investigated and applied using the Arrhenius equation. The constant value thus obtained for the activating energy showed that the active centres retained their character during immobilization. The differences between the velocities of the reaction with immobilized and with native enzyme corresponded to the different number of active centres during the reaction time. Based on these results a kinetic model of the mechanism of the studied reaction is presented which includes an initial balanced stage of the chemosorption type.     

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.     

Preparation of an immobilized two-enzyme system, beta-amylase-pullulanase, to an acrylic copolymer for the conversion of starch to maltose. II. Cocoupling of the enzymes and use in a packed bed column

β-Amylase (EC 3.2.1.2) and pullulanase (EC 3.2.1.9) have been covalently bound in a two-enzyme system to a crosslinked copolymer of acrylarmide-acrylic acid by using a water-soluble carbodiimide. The coupling yields based on the amounts of added β-amylase and pullulanase were 40% and 38%, respectively, with residual enzymic activities of 22% and 32% of those of free enzymes. A markedly increased operational stability was observed for the immobilized two-enzyme system compared to the free enzymes in solution. In order to find optimal operational conditions the influence of different pH values and temperatures on the conversion process was investigated. The action of the immobilized β-amylase-pullulanase derivative on partially hydrolyzed starch (DE 3.4–10.7) in a packed bed column was studied. Analysis of the product was performed using gas-liquid chromatography.     

Cloning and expression of the endo-1,3(4)-β-glucanase gene from Paecilomyces sp. FLH30 and characterization of the recombinant enzyme

The cDNA encoding β-1,3(4)-glucanase, named PsBg16A, from Paecilomyces sp. FLH30 was cloned, sequenced, and over expressed in Pichia pastoris, with a yield of about 61,754 U mL?1 in a 5-L fermentor. PsBg16A has an open reading frame of 951 bp encoding 316 amino acids, and the deduced amino acid sequence of PsBg16A revealed that it belongs to glycoside hydrolase family 16. The purified recombinant PsBg16A had a pH optimum at 7.0 and a temperature optimum at 70 °C, and randomly hydrolyzed barley β-glucan, lichenin, and laminarin, suggesting that it is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-glucans.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.     

Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.