Cell-associated acid β-xylosidase production by Penicillium sclerotiorum

In recent decades, β-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 °C, under stationary condition for six days. The optimum activity temperature was 60 °C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 °C, with half-life of 240 and 232 min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The β-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V_max and K_m values was 0.48 μmol PNXP min^?1 mg^?1 protein and 0.75 mM, respectively. The enzyme was xylose tolerant with a K_i of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.     

A highly-active endo-1,3-1,4-β-glucanase from thermophilic Talaromyces emersonii CBS394.64 with application potential in the brewing and feed industries

A 1245-bp endoglucanase gene of glycoside hydrolase (GH) family 7, egl7A, was cloned from the acidothermophilic fungus Talaromyces emersonii CBS394.64 and successfully expressed in Pichia pastoris. Sequence alignments indicated that Egl7A had highest identity of 62.7% at the amino acid level with the functionally characterized endoglucanase from Aspergillus terreus NIH2624. Purified recombinant Egl7A exhibited the maximum activity at pH 4.5 and 70 °C, retained stable over the pH range of 2.0–12.0 and at 65 °C, and was strongly resistant to acidic and neutral proteases, most metal ions and SDS. The enzyme exhibited the highest specific activity reported so far (11,299 U mg⁻¹) when using barley β-glucan as the substrate. Egl7A exhibited broad substrate specificity, including barley β-glucan, lichenin, CMC-Na, and xylan and had capacity to cleave cellopentaose and cellohexaose into smaller units rapidly. Under simulated mashing conditions, addition of Egl7A reduced the mash viscosity by 12.40%; when combined with a GH10 xylanase, more viscosity reduction (27.75%) was observed, which is significantly higher than that of the commercial enzyme Ultraflo XL (17.91%). All these properties make Egl7A attractive for potential applications in the feed and brewing industries.     

Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida

A novel GH10 endo-β-1,4-xylanase (XylG) gene from Streptomyces thermocarboxydus HY-15, which was isolated from the gut of Eisenia fetida, was cloned, over-expressed, and characterized. The XylG gene (1182 bp) encoded a polypeptide of 393 amino acids with a deduced molecular mass of 43,962 Da and a calculated pI of 6.74. The primary structure of XylG was 69% similar to that of Thermobifida fusca YX endo-β-1,4-xylanase. It was most active at pH 6.0 and 55 °C. The susceptibilities of xylans to XylG were as follows: oat spelt xylan > birchwood xylan > beechwood xylan. The XylG also showed high activity (474 IU/mg) toward p-nitrophenylcellobioside. Moreover, at pH 6.0 and 50 °C, the V_max and K_m values of the XylG were 127 IU/mg and 2.51 mg/ml, respectively, for oat spelt xylan and 782 IU/mg and 5.26 mM, respectively, for p-nitrophenylcellobioside. A homology model indicated that XylG folded to form a (β/α)₈-barrel with two catalytic residues of an acid/base (Glu181) and a nucleophile (Glu289). The formation of a disulfide bond between Cys321 and Cys327 were predicted by homology modeling.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

High-loading oil palm empty fruit bunch saccharification using cellulases from Trichoderma koningii MF6

Strain Trichoderma koningii D-64 was improved for enhanced cellulase production. A potential mutant MF6 was obtained and its enzymes contained filter paper cellulase (FPase), carboxymethylcellulase (CMCase), β-glucosidase and xylanase with respective activities of 2.0, 1.3, 2.0 and 3.0 folds of those for the parental strain. MF6 cellulases showed enhanced hydrolysis performance for the treated lignocellulosic biomass. Hydrolysis of treated oil palm empty fruit bunch (OPEFB), horticulture wastes (HW) and wood chips (WC) resulted in cellulose to glucose conversion of 96.3 ± 2.2%, 98.2 ± 3.0% and 81.9 ± 1.4%, respectively. The corresponding conversions of xylan to xylose were 96.9 ± 1.5%, 95.0 ± 2.2% and 76.1 ± 3.1%. Consistently, high sugar yield of 770–844 mg/g biomass was obtained for high-loading (10–16%, w/v) of OPEFB hydrolysis and sugar titer of 135.1 g/L was obtained for 16% (w/v) OPEFB loading at 96 h. In addition, MF6 enzymes alone performed equally well for high-loading OPEFB hydrolysis compared to the enzyme mixture of β-glucosidase from Aspergillus niger and cellulase from T. reesei Rut C30.     

Biochemical characterization,cloning and molecular modeling of a detergent and organic solvent-stable family 11 xylanase from the newly isolated Aspergillus niger US368 strain

New β-1,4-d-xylan xylanohydrolase (XAn11) belonging to the xylanase 11 family was purified to homogeneity from a newly soil-isolated Aspergillus niger US368 strain. The pure xylanase is a glycosylated monomer having a molecular mass of about 26 kDa. The N-terminal sequence of the purified enzyme was determined and compared to some Aspergillus xylanases N-terminal ones. The gene encoding the XAn11 was cloned and sequenced.The maximal xylanase activity was obtained at pH 5.0 and 55 °C. The XAn11 was found to be stable in a wide range of pH (3–9) and in presence of some detergents and organic solvents. A specific activity of about 805.6 U/mg or 334 U/mg was measured using birchwood xylan or oatspelt xylan as substrate, respectively. A structural explanation of the difference between experimental and theoretical molecular mass as well as the stability of the enzyme against acidic pH was proposed by molecular modeling.     

Bacteriophage phi11 lysin: Physicochemical characterization and comparison with phage phi80α lysin

Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11 (LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The objectives of the study were to investigate the stability of lysins of phages phi11 and phi80α in storage and functioning conditions, to identify optimum storage conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and electrophoresis were used to identify causes of inactivation. At 37 °C, pH 7.5 and concentration of NaCl not higher than 150 mM, LysPhi11 molecules contain a high percentage of random coils (43%). However, in spite of this the enzyme has high activity (0.4–0.8 OD_600 nm s⁻¹ mg⁻¹). In storage conditions (4 °C and 22 °C, pH 6.0–9.0, 10–500 mM NaCl) LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for LysPhi11 (4 °C, pH 6.0–7.5, 10 mM NaCl) were selected under which the time of the enzyme half-inactivation is 120–160 days. LysPhi80α stability is insufficient: at 37 °C the enzyme loses half of its activity almost immediately; at 4 °C and 22 °C the time of half-inactivation of LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent.     

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the k_cat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.     

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t_(1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol⁻¹ for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Production and characterization of a thermostable endo-type β-xylanase produced by a newly-isolated Streptomyces thermocarboxydus subspecies MW8 strain from Jeju Island

A xylanase-producing, Gram-positive, aerobic, and spore-forming bacterium was isolated from a soil sample collected from Jeju Island and was classified as a novel subspecies of Streptomyces thermocarboxydus on the basis of 16S rRNA gene sequence similarity, the results of DNA–DNA hybridization analysis, and phenotypic characteristics. The novel strain was named as S. thermocarboxydus subsp. MW8 (=KCTC29013 = DSM52054). This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 52 kDa. The NH₂-terminal sequence (Ala-Glu-Ile-Arg-Leu) was distinct from those of previously reported xylanases. The purified xylanase produced xylobiose as the end-product of birchwood xylan hydrolysis. The K_m and V_max values of the purified xylanase on birchwood xylan were 1.71 mg/ml and 357.14 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 7.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was active over broad pH ranges: 84% of the maximum activity at pH 5.0, 84–88% at pH 6.0, 88% at pH 8.0, and 75–81% (pH 9.0). These enzymatic properties may be very useful for use in bio-industrial applications.     

Purification,characterization and secondary structure elucidation of a detergent stable,halotolerant, thermoalkaline protease from Bacillus cereus SIU1

A thermoalkaline protease with a molecular weight of 22 kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the K_m, V_max and k_cat to be 1.09 mg ml^?1, 0.909 mg ml^?1 min^?1 and 3.11 s^?1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45–55 °C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, β-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0 mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a β-pleated structure and a detailed structural composition showed ～50% β-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30 min.     

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Cloning,expression, and characterization of a thermostable β-xylosidase from thermoacidophilic Alicyclobacillus sp. A4

A β-xylosidase gene (xylA4) was identified in the genome sequence of thermoacidophilic Alicyclobacillus sp. A4. The deduced amino acid sequence was highly homologous with the β-xylosidases of family 52 of the glycoside hydrolases (GH). The full-length gene consisted of 2097 bp and encoded 698 amino acids without a signal peptide. The gene product was successfully expressed in Escherichia coli with an activity of 564.9 U/mL. Recombinant XylA4 was purified by Ni²⁺-NTA affinity chromatography with a molecular mass of 78.5 kDa. The enzyme showed optimal activity at pH 6.0 and 65 °C, and remained stable over the pH range of 5.0–9.0. The thermostability of XylA4 is noteworthy, retaining almost all of the activity after 1 h incubation at 65 °C. Using p-nitrophenyl-β-d-xylopyranoside (pNPX) as the substrate, XylA4 had the highest specific activity (261.1 U/mg) and catalytic efficiency (601.5/mM/s) known so far for GH52 xylosidases. The enzyme displayed high tolerance to xylose, with a K_i value of approximately 88.7 mM. It also had synergy with xylanase XynBE18 from Paenibacillus sp. E18 in xylan degradation, releasing more xylose (up to 1.43 folds) than XynBE18 alone. Therefore, this thermostable xylose-tolerant β-xylosidase may have a great application potential in many industrial fields.     

A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

A strain of Enterobacter cloacae B5 producing β-galactosidase with transglycosylation activity was isolated from the soil. Its freeze-thawed cells synthesized galacto-oligosaccharides with a high yield of 55% from 275 g/L lactose at 50 °C for 12 h. A novel β-galactosidase capable of glycosyl transfer was purified from this strain. It was a homotetramer with molecular mass of about 442 kDa. The optimal pH and temperature for hydrolysis activity on o-nitrophenyl-β-d-galactopyranoside (oNPGal) were 6.5–10.5 and 35 °C, respectively. The enzyme showed a wide range of acceptor specificity for transglycosylation and catalyzed glycosyl transfer from oNPGal to various chemicals such as galactose, glucose, fructose, arabinose, mannose, sorbose, rhamnose, xylose, cellobiose, sucrose, trehalose, melibiose, inositol, mannitol, sorbitol and salicin, resulting in novel saccharide yields ranging from 0.8% to 23.5%. A gene encoding the enzyme was cloned and the recombinant enzyme from Escherichia coli had similar transglycosylation activity to the natural enzyme.     

Partial nitrification under limited dissolved oxygen conditions

Partial nitrification to nitrite is technically feasible and economically favourable, especially when wastewaters contained high ammonium concentrations or low C/N ratios. Partial nitrification can be obtained by selectively inhibiting nitrite-oxidizing bacteria (NOB) through appropriate regulation of the pH, temperature and dissolved oxygen (DO) concentrations. The effect of pH, DO levels and temperature on ammonia oxidation rate and nitrite accumulation was investigated in order to determine the optimal conditions for partial nitrification of synthetic wastewater with high ammonia concentration. The experiments performed at low DO levels to lower the total oxygen needed in the nitrification step, which means great saving in aeration. During the start-up stage pH and DO were set at 7.0–7.4 and 0.5 mg/l, respectively. The reactor was operated until complete partial nitrification was achieved. The effect of pH, DO on partial nitrification was studied, as pH was kept at 6.5, 7.5, 8.5, 9.5 and DO at 0.5±0.2, 1.5±0.2 and 2.5±0.2 mg/l, and temperature at 30 °C. The influence of temperature on k_a value was studied by keeping pH=7.5, DO=1.5 mg/l and temperature was controlled at 12, 20 and 30 °C, respectively. The results showed that partial nitrification to nitrite was steadily obtained and the optimal operational parameters were pH=7.5, DO=1.5 mg/l, T=30 °C based on ammonia oxidation rate and nitrite accumulation rate. The maximum k_a was achieved and to be 115.1×10⁻³ mg NH₄⁺–N (mg VSS h)⁻¹ under this condition.     

Stability of phycocyanin extracted from Spirulina sp.: Influence of temperature,pH and preservatives

Temperature and pH play an important role in the stability of phycocyanin, a natural blue colorant. Systematic investigations showed the maximum stability of phycocyanin was in the pH range 5.5–6.0. Incubation at temperatures between 47 and 64 °C caused the concentration (C_R) and half-life of phycocyanin in solution to decrease rapidly. The C_R value remained at approximately 50% after incubating for 30 min at 59 °C. After heating at 60 °C for 15 min, the C_R value of phycocyanin at pH 7.0 was maintained at around 62–70% when 20–40% glucose or sucrose was added, and the half-life increased from 19 min to 30–44 min. 2.5% sodium chloride was found to be an effective preservative for phycocyanin at pH 7.0 as a C_R value of 76% was maintained and the half-life of 67 min was increased.     

Degradation of chitin and production of bioactive materials by bioconversion of squid pens

Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications.     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.