Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228 ± 16 U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883 ± 563 U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604 U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50 °C, respectively. Activity was stable across the pH range 3.5–9.0, and the half-life was 52 min at 42 °C. K_m and V_max were calculated as 86.7 ± 5.3 mM and 928 ± 35 U/mg, and the molecular weight was approximately 65.6 kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.     

Cloning,overexpression, purification,and site-directed mutagenesis of xylitol-2-dehydrogenase from Candida albicans

Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD⁺ as a cofactor. The recombinant caXDH has a K_M of 8.8 mM and 37.7 μM using the substrate xylitol and NAD⁺, respectively, and its catalytic efficiency is 53,200 min^?1 mM^?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD⁺ to NADP⁺ in which the K_M and k_cat/K_M towards NADP⁺ are 119 μM and 26,200 min^?1 mM^?1, respectively.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

A systems level engineered E. coli capable of efficiently producing L-phenylalanine

The biosynthesis of L-phenylalanine (Phe) is one of the most complicated amino acid synthesis pathways. In this study, the engineering of Phe producer was carried out to illustrate the effectiveness of systems level engineering: (1) inactivated glucose specific phosphoenolpyruvate-carbohydrate phosphotransferase (PTS) system by inactivation of crr to moderate the glucose uptake rate to reduce overflow metabolism; (2) genetic switch on or off the expression of phe^fbr, aroG15, ydiB, aroK, and tyrB to increase the supply of precursors; (3) employed a tyrA mutant strain to reduce carbon diversion and to result in non-growing cells; (4) enhanced the efflux of Phe by overexpressing yddG to shift equilibrium towards Phe synthesis and to release the feedback regulation in Phe synthesis. The mutants in PTS were firstly compared and a crr⁻ mutant was firstly screened. The mutant AroG15 was demonstrated to a thermostable mutant. The strains expressing yddG excreted Phe into the medium at higher rate and less intracellular Phe accumulated. By systems level engineering, an engineered Phe producer achieved 47.0 g/L Phe with a yield of 0.252 g/g which was the highest under the non-optimized fermentation condition.     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.     

Purification and characterization of aminopeptidase N from chicken intestine with potential application in debittering

An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken (Gallus gallus) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be ∼180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 °C and pH 6.0. The enzyme preferentially hydrolyzed Leu-β-NA (K_m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (1 mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed.     

Domain replacement to elucidate the role of B domain in CGTase thermostability and activity

The B domain of CGTase has been generally accepted as a domain involved in thermostability. However, limited work has been performed in which entire B domain is substituted with the thermostable counterpart. Using overlap extension PCR, we replaced the B domain of a variant of CGTase Bacillus sp. G1 by six other B domains from thermostable CGTases. Likely due to distortion in the substrate-binding cleft adjacent to the active site, variants with the domain replacements from Thermoanaerobacter, Thermococcus, Thermococcus kodakarensis, Anaerobranca gottschalkii and Pyrococcus furiosus completely lost their catalytic function. A mutant designated Cgt_ET1 with a domain replacement from a Bacillus stearopthermophilus ET1 CGTase was the only variant that retained activity after domain exchange. Both the parental enzyme and the mutant Cgt_ET1 had an identical optimum temperature at 60 °C. The activity half-life was 22 min for the parental CGTase, whereas a marked increase to 57 min was observed for the mutant. Further mutagenesis on Cgt_ET1 was performed at residue 188 by replacing a Phe residue with Tyr. The mutant Cgt_ET1_F188Y displayed a decreased activity half-life of 28 min. Both mutants exhibited a better cyclodextrin-forming ability and a faster turnover rate (k_cat) than the parental CGTase.     

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region (“NB loop”). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called “I-loop” region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K_m with PPi (up to 33 mM), whereas Ncut deletions had greatly increased K_m with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K_m values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.     

Active-site mutations improved the transglycosylation activity of Stenotrophomonas maltophilia chitinase A

Transglycosylation (TG) by family 18 chitinases is of special interest due to the many biological applications of long-chain chitooligosaccharides (CHOS). In the current study, the TG activity of chitinase A from Stenotrophomonas maltophilia (StmChiA) was improved through structure-guided mutations within and around the active site. Three independent mutants were created, targeting Trp residues from the − 3 and − 1 subsites and the central catalytic Asp from the DxDxE motif of StmChiA. The former was replaced with Ala and the latter with Asn. Changes in the hydrolytic and TG activities of the enzymes were assessed by monitoring the product profile of each mutant by high-performance liquid chromatography. All three mutants showed increased TG activity. Increased in the higher TG activity of mutant W306A was accompanied by increased hydrolysis. However, this mutant also accumulated substantial amounts of TG products during the first 15–30 min of the reaction. In contrast, mutants D464N and W679A showed reduced hydrolysis, which was accompanied by the gradual accumulation of TG products up to 12 h. Molecular docking studies with chitohexaose showed that the side chains of Trp residues mediate stacking interactions with sugar residues from the − 3 and − 1 subsites, indicating the importance of these residues in the enzymatic activity of StmChiA. Overall, mutants of the glycon-binding site (W306A and W679A) appear to produce long-chain CHOS more efficiently than the catalytic mutant D464N.     

Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content.Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110 kDa has an optimal pH of 7.0 and a pI of 5.6. It splits β-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl β-naphthylamide (Leu βNA) is the best substrate with the highest hydrolytic coefficiency followed by Met βNA = Arg βNA = Lys βNA > Ala βNA > Tyr βNA > Phe βNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K_m 82 μM and a k_cat of 1.08 s⁻¹, and Met-enkephalin with a K_m of 106 μM and a k_cat of 2.6 s⁻¹. The puromycin-sensitive enzyme is most susceptible to amastatin with an IC₅₀ of 0.05 μM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1 A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (∼300 s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1 A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1 A-, GSTP1-1B-, and GST1-1D-expressing cells OH generation resulted (after 36 h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (K_D=51.0 vs 57.0 nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (K_D=101.0 (94.0) nM). In silico modeling of the GSTP1-1–Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress.     

Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)_2–4, were applied. The k_cat/K_m values for (GlcNAc)_2–4 are 239.8, 111.3 and 79.8 s^?1 mM^?1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.     

Integrated biooxidation and acid dehydration process for monohydroxylation of aromatics

We examined the performance of an integrated biooxidation and acid dehydration process for aromatic monohydroxylation using the production of o-cresol from toluene as the model conversion. A toluene cis-glycol dehydrogenase gene (todD)-disrupted mutant of Pseudomonas putida T-57 was employed as the whole cell biocatalyst for oxidizing toluene to toluene cis-glycol (TCG). After bioconversion of toluene to TCG, o-cresol was produced by adding HCl to the culture medium, since it was found that TCG became unstable at a low pH and underwent spontaneous dehydration. When the todD-disrupted mutant cells were grown in a two-liquid-phase culture system with oleyl alcohol as the organic solvent, this integrated process produced 40 g l⁻¹ of o-cresol in the organic solvent phase at 30 h. The total concentration of o-cresol in the two-liquid-phase culture reached 6.6 g l⁻¹ at 30 h, which was approximately four-fold greater than that in the single-liquid-phase culture.     

Enhancing the enzymatic activity of the endochitinase by the directed evolution and its enzymatic property evaluation

Endochitinase has an important application in the biological treatment of chitin, the second most abundant and renewable resource in nature. In order to enhance its activity, a double mutant strain MECH-Y185/S226 was obtained by the directed evolution using the error-prone PCR with the mature endochitinase cDNA from Trichoderma viride as the template. Compared to those of the primitive strain MECH, endochitinase activities of the mutant one were 1.8-fold higher towards 4-nitrophenyl-N-acetyl-β-d-glucosaminide and 3.5-fold higher towards the colloidal chitin. Sequence alignments indicated that 9 nucleotides and 2 amino acids (Y185F and S226P) were mutant. The SDS–PAGE analysis showed that a single band with an estimated molecular weight of 46 kDa was obtained when the Ech42-Y185/S226 was purified sequentially by ammonium sulfate precipitation, DE52 anion-exchanging column chromatography and Sephadex G-100 column chromatography. Kinetic parameters K_m and V_max of the Ech42-Y185/S226 were 0.25 ± 0.02 mmol/l and 4.59 ± 0.32 μmol/l min, respectively. The analysis of enzymatic properties showed that the Ech42-Y185/S226 had a higher thermal stability at higher temperatures and a higher pH stability within a wider pH range than the Ech42. Observed activities of the Ech42-Y185/S226 are the highest in the presence of Mg²⁺ and the lowest in the presence of Zn²⁺.     

Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign

l-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel l-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7 IU/mg, which is higher than native (rBliA) (407.65 IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with K_m value of 0.42 mM and V_max of 2778.9 μmol min⁻¹.     

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression,purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V_max and K_m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.     

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD^D485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad^D485G) ABE products resulted in a blend with nearly 50 wt%≤C₉ products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L⁻¹ while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C₁₁ which amounts to 9.8 g L⁻¹ of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.     

Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei), Finizym (from Aspergillus niger), Ultraflo L (from Humicola insolens), and Viscozyme L (from Aspergillus aculeatus) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60 °C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60 °C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50 °C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α-l-arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations.     

Glucose and xylose stimulation of a β-glucosidase from the thermophilic fungus Humicola insolens: A kinetic and biophysical study

β-Glucosidases activated by glucose and xylose are uncommon yet intriguing enzymes that may enhance cellulose saccharification efficiency, and are of interest for application in bioethanol production processes. The molecular mechanisms of activation are completely unknown, and the aim of this study was the kinetic and biophysical characterization of the stimulation of a β-glucosidase from Humicola insolens by glucose and xylose. The effects of the monosaccharides were concentration dependent, where in a stimulatory range (0.1–50 mmol L⁻¹), the activity increased up to 2-fold; in a stimulatory-inhibitory range (50–450 mmol L⁻¹ glucose or 50–730 mmol L⁻¹ xylose), the enzyme continued to be stimulated, but the activity was lower than maximal. Above 450 mmol L⁻¹ glucose or 730 mmol L⁻¹ xylose, increasing inhibition occurred. Dynamic light scattering confirmed that the enzyme is monomeric (54 kDa) and kinetic, intrinsic tryptophan fluorescence emission and far ultraviolet circular dichroism analyses indicated that the enzyme possesses a catalytic site (CS) and a modulator binding site (MS). Glucose or xylose binding to the MS induces conformational changes that stimulate the catalytic activity at the CS. Glucose and xylose may compete with the substrate for the CS while the substrate competes with the monosaccharides for binding to the MS. The stimulation of the enzymatic activity by glucose and xylose, which compete for the same sites on the enzyme molecule, is not synergistic. These data reveal allosteric interactions between the MS and the CS in H. insolens β-glucosidase that result in fine modulation of the catalytic activity by the monosaccharides. A kinetic model was developed that accurately described the experimental data for enzyme stimulation by glucose and/or xylose. Understanding the regulatory mechanisms of the enzyme activity, with the aid of kinetic models, may be useful for the application of the enzyme in cellulose hydrolysis processes.