Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO₂ hydration: PfCAdom showed a k_cat of 3.8 × 10⁵ s⁻¹ and k_cat/K_m of 7.2 × 10⁷ M⁻¹ × s⁻¹, whereas PfCA showed a lower activity compared to PfCAdom, with a k_cat of 1.4 × 10⁵ s⁻¹ and k_cat/K_m of 5.4 × 10⁶ M⁻¹ × s⁻¹. PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed K_Is in the range of 9–68 μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with K_Is in the range of 0.53–0.97 mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action.     

pH-rate profiles of l-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in l-xylulose production

l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD⁺, catalyzed by HjLAD, was studied within the pH range of 7.0–9.5 at 25 °C. The turnover number (k_cat) and the catalytic efficiency (k_cat/K_m) were 4200 min⁻¹ and 290 mM⁻¹ min⁻¹, respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.     

Identification of a marine NADPH-dependent aldehyde reductase for chemoselective reduction of aldehydes

A putative aldehyde reductase gene from Oceanospirillum sp. MED92 was overexpressed in Escherichia coli. The recombinant protein (OsAR) was characterized as a monomeric NADPH-dependent aldehyde reductase. The kinetic parameters K_m and k_cat of OsAR were 0.89 ± 0.08 mM and 11.07 ± 0.99 s⁻¹ for benzaldehyde, 0.04 ± 0.01 mM and 6.05 ± 1.56 s⁻¹ for NADPH, respectively. This enzyme exhibited high activity toward a variety of aromatic and aliphatic aldehydes, but no activity toward ketones. As such, it catalyzed the chemoselective reduction of aldehydes in the presence of ketones, as demonstrated by the reduction of 4-acetylbenzaldehyde or the mixture of hexanal and 2-nonanone, showing the application potential of this marine enzyme in such selective reduction of synthetic importance.     

Anion inhibition study of the β-class carbonic anhydrase (PgiCAb) from the oral pathogen Porphyromonas gingivalis

The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the β-class (PgiCAb) and another one to the γ-class (PgiCA). This last enzyme has been characterized earlier for its inhibition profile with various classes of CA inhibitors, such as the sulfonamides and anions, whereas for PgiCAb such data were not yet reported. Here we show that PgiCAb has a good catalytic activity for the CO₂ hydration reaction, with k_cat 2.8 × 10⁵ s⁻¹ and k_cat/K_m of 1.5 × 10⁷ M⁻¹ × s⁻¹, being inhibited by cyanate and diethyldithiocarbamate in the submillimolar range (K_Is of 0.23–0.76 mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (K_Is of 60–78 μM). The anion inhibition profile of the two P. gingivalis enzymes is very different. Identification of selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available.     

Cloning,characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea

We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO₂ hydration to bicarbonate and protons, with the following kinetic parameters: k_cat of 6.0 × 10⁵ s⁻¹ and a k_cat/K_m of 4.7 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a K_I of 502 nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed K_Is in the range of 1.4–4.4 mM. Diethyldithiocarbamate was a better inhibitor (K_I of 0.58 mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with K_Is ranging between 8 and 38 μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.     

Isolation and divalent-metal activation of a β-xylosidase,RUM630-BX

The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (k_cat) by divalent metals Mg²⁺, Mn²⁺ and Co²⁺ but not by Ca²⁺, Ni²⁺, and Zn²⁺. Activation of RUM630-BX by Mg²⁺ (t_0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (k_cat/K_m) of 299 s⁻¹ mM⁻¹ on xylotetraose being the highest reported.     

Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes

A yellow laccase from the culture filtrate of Trametes hirsuta MTCC-1171 has been purified. The purification methods involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 55.0 kDa. Using 2,6-dimethoxyphenol, 2,2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as the substrates, the K_m, k_cat and k_cat/K_m values of the laccase were found to be 420 μM, 13.04 s⁻¹, 3.11 × 10⁴ M⁻¹ s⁻¹, 225 μM, 13.03 s⁻¹, 1.3 × 10⁵ M⁻¹ s⁻¹ and 100 μM, 13.04 s⁻¹, 5.8 × 10⁴ M⁻¹ s⁻¹, respectively. The pH and temperature optima were 4.5 and 60 °C, respectively while pH and temperature stabilities were pH 4.5 and 50 °C. The activation energy for thermal denaturation of the enzyme was 18.6 kJ/mol/K. The purified laccase has yellow colour and does not show absorption band around 610 nm like blue laccases. The purified laccase transforms toluene, 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator molecules in high yields.     

Gene cloning and expression of a detergent stable alkaline protease from Aspergillus clavatus ES1

The genes, cDNA alpES1 and alpES1, encoding Aspergillus clavatus ES1 alkaline protease were amplified from complementary DNA (cDNA) and genomic DNA, respectively, cloned in pCR^®II-TOPO plasmid and then sequenced. Sequence analysis of the cDNA alpES1 gene revealed an open reading frame (ORF) of 1212 bp encoding a pre–pro-protein of 403 amino acid residues consisting of a 21-aa signal peptide, a 100-aa pro-peptide and a 282-aa mature protein with a calculated molecular weight of 28.5 kDa. Compared to the cDNA alpES1 gene, the alpES1 gene contained three introns, which had 53, 57 and 54 bp, respectively. The cDNA alpES1 gene was then sub-cloned in pET-30b(+) and expressed in Escherichia coli BL21 (λDE3). The purified recombinant protease had a molecular weight of about 32 kDa estimated by SDS-PAGE. Kinetic parameters, K_m and k_cat values of the recombinant AlpES1 for casein, were 0.23 mM and 12.38 min⁻¹, respectively. The catalytic efficiency (k_cat/K_m) was 53.82 min⁻¹ mM⁻¹.     

Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae

Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration–dehydration reaction: CO₂ + H₂O ⇋ HCO₃⁻ + H⁺. Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO₂ hydration, with k_cat values ranging between (3.4 − 8.23) × 10⁵ s⁻¹ and k_cat/K_M of (4.1 − 7.0) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging between 44 and 91 μM.     

Anion inhibition studies of two new β-carbonic anhydrases from the bacterial pathogen Legionella pneumophila

We investigated the cloning, catalytic activity and anion inhibition of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Legionella pneumophila. Two such enzymes, lpCA1 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO₂ hydration, with k_cat values in the range of (3.4–8.3) × 10⁵ s⁻¹ and k_cat/K_M values of (4.7–8.5) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated to identify inhibitors of these enzymes. Perchlorate and tetrafluoroborate were not acting as inhibitors (K_I >200 mM), whereas sulfate was a very weak inhibitor for both lpCA1 and lpCA2 (K_I values of 77.9–96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging from 6 to 94 μM. The most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_I values ranging from 2 to 13 μM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action.     

Sulfonamide inhibition studies of two β-carbonic anhydrases from the bacterial pathogen Legionella pneumophila

Two β-carbonic anhydrases (CAs, EC 4.2.1.1) were identified, cloned and purified in the pathogenic bacterium Legionella pneumophila, denominated LpCA1 and LpCA2. They efficiently catalyze CO₂ hydration to bicarbonate and protons, with k_cat in the range of (3.4–8.3) × 10⁵ s⁻¹ and k_cat/K_m of (4.7–8.5) × 10⁷ M⁻¹ s⁻¹, and are inhibited by sulfonamides and sulfamates. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide(K_Is in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide and dichlorophenamide (K_Is in the range of 25.2–88.5 nM). As these enzymes may be involved in pH regulation in the phagosome during Legionella infection, their inhibition may lead to antibacterials with a novel mechanism of action.     

Anion inhibition study of the β-carbonic anhydrase (CahB1) from the cyanobacterium Coleofasciculus chthonoplastes (ex-Microcoleus chthonoplastes)

We investigated the catalytic activity and inhibition of the β-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO₂ hydration, with a k_cat of 2.4 × 10⁵ s⁻¹ and a k_cat/K_m of 6.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (K_Is >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with K_Is of 29.9–48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar–millimolar inhibitors (K_Is in the range of 0.15–0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 8–75 μM, whereas acetazolamide inhibited the enzyme with a K_I of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO₂ fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO₂ capture processes.     

Characterization of a novel thermostable l-rhamnose isomerase from Thermobacillus composti KWC4 and its application for production of d-allose

d-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn²⁺ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (K_m), turnover number (k_cat) and catalytic efficiency (k_cat/K_m) for l-rhamnose were 33.8 mM, 1189.8 min⁻¹ and 35.2 min⁻¹ mM⁻¹, respectively. At a higher temperature, Mn²⁺ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (T_m) of T. composti L-RI was increased by 3 °C in presence of Mn²⁺. Although the T. composti L-RI displayed the optimum substrate as l-rhamnose, it could also effectively catalyze the isomerization between d-allulose and d-allose. When the reaction reached equilibrium, the sole product d-allose was produced from D-alluose by T. composti L-RI.     

Study into the kinetic properties and surface attachment of a thermostable adenylate kinase

A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (k_cat/K_M) increased from 8.45±3.04 mM^?1 s^?1 in solution to 32.23±3.20 or 24.46±4.41 mM^?1 s^?1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.     

Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato,Ipomoea batatas (L.) Lam.

Ortho-hydroxylation of cinnamates is a key step in coumarin biosynthesis in plants. Ortho-hydroxylated cinnamates undergo trans/cis isomerization of the side-chain and then lactonization to form coumarins. Sweet potato [Ipomoea batatas (L.) Lam.] accumulates umbelliferone and scopoletin after biotic and abiotic stresses. To elucidate molecular aspects of ortho-hydroxylation involved in umbelliferone formation in sweet potato, isolation and characterization of cDNAs encoding 2-oxoglutarate-dependent dioxygenases (2OGD) was performed from sweet potato tubers treated with a chitosan elicitor. Five cDNAs (designated as Ib) encoding a protein of 358 amino acid residues were cloned, and these were categorized into two groups, Ib1 and Ib2, based on their amino acid sequences. Whether the recombinant Ib proteins had any enzymatic activity toward cinnamates was examined. Ib1 proteins exhibited ortho-hydroxylation activity toward feruloyl coenzyme A (CoA) to form scopoletin (K_m = ∼10 μM, k_cat = ∼2.7 s⁻¹). By contrast, Ib2 proteins catalyzed ortho-hydroxylation of feruloyl-CoA (K_m = 7.3–14.0 μM, k_cat = 0.28–0.55 s⁻¹) and also of p-coumaroyl-CoA (K_m = 6.1–15.2 μM, k_cat = 0.28–0.64 s⁻¹) to form scopoletin and umbelliferone, respectively. Fungal and chitosan treatments increased levels of umbelliferone and its glucoside (skimmin) in the tubers, and expression of the Ib2 gene was induced concomitantly.     

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ～456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T_1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol^?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg²⁺ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) for sodium phytate were 83 nmol mg^?1 s^?1 and 0.156 mM, respectively. The catalytic turnover number (K_cat) and catalytic efficiency (K_cat/K_m) of phytase were 37.8 s^?1 and 2.4 × 10⁵ M^?1 s^?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.     

Effects of flooding on photosynthesis and root respiration in saltcedar (Tamarix ramosissima), an invasive riparian shrub

The introduced shrub Tamarix ramosissima invades riparian zones, but loses competitiveness under flooding. Metabolic effects of flooding could be important for T. ramosissima, but have not been previously investigated. Photosynthesis rates, stomatal conductance, internal (intercellular) CO₂, transpiration, and root alcohol dehydrogenase (ADH) activity were compared in T. ramosissima across soil types and under drained and flooded conditions in a greenhouse. Photosynthesis at 1500 μmol quanta m⁻² s⁻¹ (A₁₅₀₀) in flooded plants ranged from 2.3 to 6.2 μmol CO₂ m⁻² s⁻¹ during the first week, but A₁₅₀₀ increased to 6.4–12.7 μmol CO₂ m⁻² s⁻¹ by the third week of flooding. Stomatal conductance (g_s) at 1500 μmol quanta m⁻² s⁻¹ also decreased initially during flooding, where g_s was 0.018 to 0.099 mol H₂O m⁻² s⁻¹ during the first week, but g_s increased to 0.113–0.248 mol H₂O m⁻² s⁻¹ by the third week of flooding. However, photosynthesis in flooded plants was reduced by non-stomatal limitations, and subsequent increases indicate metabolic acclimation to flooding. Root ADH activities were higher in flooded plants compared to drained plants, indicating oxygen stress. Lower photosynthesis and greater oxygen stress could account for the susceptibility of T. ramosissima at the onset of flooding. Soil type had no effect on photosynthesis or on root ADH activity. In the field, stomatal conductance, leaf water potential, transpiration, and leaf δ¹³C were compared between T. ramosissima and other flooded species. T. ramosissima had lower stomatal conductance and water potential compared to Populus deltoides and Phragmites australis. Differences in physiological responses for T. ramosissima could become important for ecological concerns.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 °C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (k_cat/K_m = 18.5 s^?1 μM^?1) and 2,6-dimethoxyphenol (k_cat/K_m = 13.9 s^?1 μM^?1) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield.