Cloning, Expression, and Enzymatic Characterization of Isocitrate Dehydrogenase from Helicobacter pylori

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k _cat of 87 s^?1, K _m of 124 μM and k _cat/K _m of 7 × 10⁵ M^?1s^?1 toward isocitrate, k _cat of 80 s^?1, K _m of 176 μM and k _cat/K _m of 4.5 × 10⁵ M^?1s^?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.     

GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509

A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K _m and k _cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s^?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K _m and k _cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s^?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K _m and k _cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s^?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.     

Cloning,over-expression and characterization of a thermo-tolerant xylanase from Thermotoga thermarum

The xyn10B gene, encoding the endo-1,4-β-xylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent K _m , k _cat and k _cat /K _m values of the xylanase for beechwood xylan were 1.8 mg ml^?1, 520 s^?1 and 289 ml mg^?1 s^?1, respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis.     

A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids

Leukotriene A₄ hydrolase (LTA4H––EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA₄ through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k _cat/K _m values). Among them, the benzyl ester of aspartic acid exhibited a k _cat/K _m value that was more than two orders of magnitude higher (1.75 × 10⁵ M^?1 s^?1) as compared to l-Arg (1.5 × 10³ M^?1 s^?1). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.     

Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation

A codon-optimized Escherichia coli appA phytase gene was synthesized and expressed in Pichia pastoris. Two residue substitutions (Q258N, Q349N) were sequentially introduced to enhance its glycosylation activity. Secretion of appA-Q258N/Q349N was approx. 0.3 mg ml^?1 and enzyme activity reached 1,030 U ml^?1. Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg^?1 with an MW of approx. 53 kDa. Compared with appA-WT, appA-Q258N/Q349N showed over 40 % enhancement in thermostability (85 °C for 10 min) and 4–5 °C increases in the melting temperatures (T_m). The K_m and K_cat of appA-Q258N/Q349N were 0.43 mM and 3,058 s^?1, respectively, which are similar with that of appA-WT. The mutant appA-Q258N/Q349N obtained in this study could be used for the large-scale commercial production of phytase.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k _cat and k _cat/K _m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K _i values for d-glucose (83.3–357 mM) and d-xylose (15.6–70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K _t ^0.5?～?45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k _cat and k _cat/K _m on natural substrates xylobiose (407 s^?1, 138 s^?1?mM^?1), xylotriose (235 s^?1, 80.8 s^?1?mM^?1), and xylotetraose (146 s^?1, 32.6 s^?1?mM^?1).     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

6-Aminoquinoline as a fluorogenic leaving group in peptide cleavage reactions: A new fluorogenic substrate for chymotrypsin

A new fluorogenic substrate capable of measuring the amidolytic activity of chymotrypsin and based upon the enzyme-catalyzed release of a highly fluorescent aromatic amine, 6-aminoquinoline, was prepared. The substrate, 6-(N-glutaryl-l-phenylalanylamido)quinoline, was found to have at pH 8.0 and 25°C K_m = 1.77 mm and k_cat = 1.4 × 10^?1 s^?1. The aminoquinoline is a unique leaving group in that its appearance can be measured fluorometrically at its excitation and emission maxima, while, under these conditions, fluorescence associated with unhydrolyzed substrate is negligible.     

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex

The latex from Vasconcellea quercifolia (“oak leaved papaya”), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K _m = 0.454 ± 0.046 mM, k _cat = 1.57 ± 0.07 s^?1, k _cat/K _m = 3.46 × 10³ ± 14 s^?1 M^?1). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the “papain-like” family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P. pastoris and its applicability in fractionating allergenic glycinin from soy protein

The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K _m, V _max, K _cat, and K _cat/K _m of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg^?1 s^?1, 65,655 ± 10.92 s^?1, and 328.3 ± 3.12 μM^?1 s^?1, respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.     

Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K_m and V_max values of 0.25 mM and 5.35 μkat.mg^?1 enzyme protein. Thus, the enzyme had a very high (292,000 M^?1.s^?1) catalytic efficiency (K_cat/K_m). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol^?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K_i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.     

A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase

A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0–10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K _m value towards beechwood xylan was 0.548 mg ml^?1, and the k _cat/K _m ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg^?1 s^?1 at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries.