Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca

Glucose isomerase (GIase) catalyzes the isomerization of d-glucose to d-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5–10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K _m and K _cat values of the enzyme are 197 mM and 1,688 min^?1, respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.     

Catalytic properties of endo-1,3-β-D-glucanase from the Vietnamese edible mussel Perna viridis

A β-1,3-glucanase with a molecular mass of 33 kDa was isolated in the homogeneous state from a crystalline stalk of the commercially available Vietnamese edible mussel Perna viridis. It hydrolyzes β-1,3-bonds in glucans and is capable of catalyzing the transglycosylation reaction. The β-1,3-glucanase has a K _m value of 0.3 mg/ml for the hydrolysis of laminaran and shows a maximum activity in the pH range from 4 to 6.5 and at 45°C. Its half-inactivation time is 180 min at 45°C and 20 min at 50°C. The enzyme was ascribed to glucan-endo-(1 → 3)-β-D-glucosidases (EC 3.2.1.39). The enzyme could be used in the structure determination of β-1,3-glucans and enzymatic synthesis of new carbohydrate-containing compounds.     

Purification and properties of Bacillus coagulans cyclomaltodextrin glucanotransferase

Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans, was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca²⁺ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70°C. It did not lose its activity by heat treatment at 70°C for 10 min in the presence of CaCl₂ in the pH range of 5.5∼9.5, and by incubation in the pH range of 5.0∼10.5 at 4°C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50°C, and the ratio of α-: β-: γ-cyclodextrin produced was 8.1:8.9:1.0 B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0–6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn²⁺. The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K _m, V _max, K _cat, and K _cat/K _m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.     

Purification and characterization of an extremely stable glucose isomerase from Geobacillus thermodenitrificans TH2

The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The K _m and V _max values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 μmol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.     

Purification and Characterization of an Extracellular Low Temperature-Active and Alkaline Stable Peptidase from Psychrotrophic Acinetobacter sp. MN 12 MTCC (10786)

An extracellular low temperature-active alkaline stable peptidase from Acinetobacter sp. MN 12 was purified to homogeneity with a purification fold of 9.8. The enzyme exhibited specific activity of 6,540 U/mg protein, with an apparent molecular weight of 35 kDa. The purified enzyme was active over broad range of temperature from 4 to 60 °C with optimum activity at 40 °C. The enzyme retained more than 75 % of activity over a broad range of pH (7.0–11.0) with optimum activity at pH 9.0. The purified peptidase was strongly inhibited by phenylmethylsulfonyl fluoride, giving an indication of serine type. The K _m and V _max for casein and gelatin were 0.3529, 2.03 mg/ml and 294.11, 384.61 μg/ml/min respectively. The peptidase was compatible with surfactants, oxidizing agents and commercial detergents, and effectively removed dried blood stains on cotton fabrics at low temperature ranging from 15 to 35 °C.     

Characterization of an indican-hydrolyzing enzyme from Sinorhizobium meliloti

A novel β-glucosidase capable of hydrolyzing indican to indigo was mined and isolated from Sinorhizobium meliloti using a systematic approach. The corresponding gene was amplified by PCR and overexpressed in the soluble fraction as an MBP fusion protein. The resulting enzyme easily purified to apparent homogeneity via a consecutive step in the affinity column. The recombinant enzyme was determined to be a monomer with a calculated molecular mass of 52 kDa and showed the maximum activity for indican at pH 7.0 and 45 °C. The kinetic parameters for indican, K_M and V_max, were determined to be 0.97 mM and 355.6 μM/min/mg protein, respectively, at pH 7.0 and 35 °C. Additionally, this enzyme hydrolyzed both the β-(1-4)- and β-(1-6)-glucosidic bonds and revealed a minor activity against α-d-glucosides. Furthermore, the enzyme was severely inhibited by DTT, indicating a possibility that the oxidation of amino acids could play a crucial role in the activity of the enzyme.     

Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on β-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K–T307I were approx. 140, 190 and 200%, respectively, of wild type β-agarase (661 U/mg) at 40°C. All three mutant enzymes were stable up to 50°C and E99K–T307I had the highest thermostability. The melting temperature (T _m) of E99K–T307I, determined by CD spectra, was increased by 5.2°C over that of the wild-type enzyme (54.6°C). Activities of both the wild-type and E99K–T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl₂. The E99K–T307I enzyme was stable at 55°C with 1 mM CaCl₂, reaching 260% of the activity the wild-type enzyme held at 40°C without CaCl₂.     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.     

Stepwise synthesis of methyl 4-O-[3-O-(β-d-xylopyranosyl)-β-d-xylopyranosyl]-β-d-xylopyranoside

The general properties and specificity of a dextran α-(1→2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5–6.5, and 45°, respectively. The enzyme was stable up to 40° on heating for 10 min, and over a pH range of 6.5–9.0 on incubation at 4° for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1→2)-α-d-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces d-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the K_m value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. The debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran α-(1→2)-debranching enzyme, and its systematic name is also discussed.     

Kinetic modeling of thermal inactivation of the Bacillus sp. protease P7

Data from thermal stability of a keratinolytic protease produced by the Amazon isolate Bacillus sp. P7 was fitted to various mathematical models. Kinetic modeling showed that Weibull distribution was the best equation to describe the residual activity of protease P7 after heat treatment. The effects of temperature on equation parameters and on characteristics of the inactivation curves were evaluated. As expected, faster inactivation was observed at higher temperatures. The critical temperature to accelerate protease decomposition was about 70 °C. The reliable life (t _R) of the enzyme, analogous to the D value, ranged from 1,824 to 8 min at 45–65 °C. Within these temperatures, an increase of 8.81 °C was needed to lower enzyme t _R in one-log unit. Protease P7 is a potentially useful biocatalyst for various industrial bioprocesses, and therefore, kinetic modeling of thermal inactivation addresses an important topic aiming enzyme characterization and applications.     

Purification and Characterization of a Novel (<Emphasis Type="Italic">R</Emphasis>)-1-Phenylethanol Dehydrogenase from <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. NUST506

A novel (R)-1-phenylethanol dehydrogenase was successfully purified from Lysinibacillus sp. NUST506 by preparative polyacrylamide gel electrophoresis. The enzyme is a NAD+-dependent oxidoreductase. The molecular weight of the (R)-1-phenylethanol dehydrogenase measured by SDS-PAGE was about 28 kDa. Furthermore, the optimal reaction conditions for the oxidative reaction were 70°C and pH 9.5 and for the reductive reaction were 65°C and pH 6.5. Under the optimal conditions, the K_M and k_cat values with (R)-1-phenylethanol as a substrate were found to be 0.78 mM and 123 s^–1 and with acetophenone they were 0.56 mM and 125 s^–1, respectively. The (R)-1-phenylethanol dehydrogenase became more stable at pH 9.5 in comparison with pH 5.0 and high stability was noticed at 4 and 37°C. Properties of the enzyme place it as a promising candidate for industrial applications.     

Properties of NAD (P) H-linked aldose reductase from Crytococcus lactativorus

A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The K_m^app value for d-xylose was 8.6 mM and the V_max^app was 20.8 units/mg NADH was used as a coenzyme. The K_m^app values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate.     

Purification and properties of mycodextranase from Bacillus circulans NHB-1

A Gram-positive bacterium, Bacillus circulans, isolated from soil was found to produce an enzyme hydrolyzing nigeran (mycodextran, alternating α-1,3- and α-1,4-linked glucan). The molecular weight of the purified enzyme was 120,000 and its isoelectric point was 8.30. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 7.0 and up to 50°C. The K_m (mg/ml) for nigeran was 1.37. The enzyme specifically hydrolyzed the nigeran into nigerose and nigeran tetrasaccharide by an endo-type action, indicating that it is a mycodextranase (EC 3.2.1.61) cleaving only the α-1,4-glucosidic linkages in nigeran. The N-terminal amino acid sequence of the purified enzyme of B. circulans (APTVYEAESAAKTGGV) was different from that of the mycodexstranase purified from Streptomyces sp. J-13-3 (XDPGDPTDPDPSGVGATLPF).     

Encapsulation in a sol–gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue

Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t _1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K _m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V _max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.     

Pressure effects on the association of the and 2 subunits of tryptophan synthetase from Escherichia coli and Salmonella typhimurium

Sucrose density gradient centrifugation was employed to study the association of the α and β₂ subunits of the enzyme tryptophan synthetase from Escherichia coli and Salmonella typhimurium. In both cases, the fully associated enzyme (α₂β₂) showed a sedimentation coefficient of 6.4 S, in agreement with the values reported by other workers for the E. coli enzyme. The substrate, l-serine, and the cofactor, pyridoxal phosphate, were required for complex formation in both cases. Generation of moderately high pressures by increasing the centrifuge speed from 39,000 rpm to 50,000 rpm was found to interfere with complex formation in both species at 5 °C. This effect was reversed by a temperature increase from 5 °C to 20 °C or by low concentrations of a nonpolar solvent, ethanol, at 5 °C. These results indicate that hydrophobic bonding plays an important role in the formation of the active tryptophan synthetase α₂β₂ complex. Monovalent and divalent cations also interfered with the formation of the α₂β₂ complex, indicating the possibility that ionic bonds are also involved.