Purification and Characterization of a Bacterial Phytase Whose Properties Make it Exceptionally Useful as a Feed Supplement

A periplasmatic phytase from a bacterium isolated from Malaysian waste water was purified about 173-fold to apparent homogeneity with a recovery of 10% referred to the phytase activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 65°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K _M = 0.15 mmol/l and k _cat = 1164 s⁻¹ at pH 4.5 and 37°C. The purified enzyme was shown to be highly specific. Among the phosphorylated compounds tested, phytate was the only one which was significantly hydrolysed. Some properties such as considerable activity below pH 3.0, thermal stability and resistance to pepsin make the enzyme attractive for an application as a feed supplement.     

Protective Effect of Some Anions on the Heat-Denaturation of Ovotransferrin

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 °C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H₂O₂, 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P₁ position, in descending order. The k _cat, K _m and k _cat/K _m values for Z-Val-Lys-Met-MCA were 16.8±0.14 min^?1, 5.1±0.28 μM, and 3.3±0.28 μM^?1 min^?1 respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.     

Purification and Characterization of a Cathepsin L-Like Enzyme from the Body Wall of the Sea Cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS–PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine

7-amido-4-methylcoumarin with K _m (69.92 μM) and k _cat (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 °C. It showed thermal stability below 40 °C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k_cat/K_M)_Glc/(k_cat/K_M)_Gal = 172], the variant oxidizes both sugars equally well [(k_cat/K_M)_Glc/(k_cat/K_M)_Gal = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K_M values and approximately ten-fold higher k_cat values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60°C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D -glucose/D -galactose mixtures at both 30 and 50°C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 Å resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.     

Characterization of an apo-carotenoid 13,14-dioxygenase from Novosphingobium aromaticivorans that converts β-apo-8′-carotenal to β-apo-13-carotenone

A putative carotenoid oxygenase from Novosphingobium aromaticivorans was purified with a specific activity of 0.8?U/mg by His-Trap affinity chromatography. The native enzyme was estimated to be a 52?kDa monomer. Enzyme activity for β-apo-8′-carotenal was maximal at pH 8.0 and 45?°C, with a half life of 15.3?h, K _m of 21?μM, and k _cat of 25?l/min. The enzyme exhibited cleavage activity only for carotenoids containing one β-ionone ring and its catalytic efficiency (k _cat/K _m) followed the order β-apo-8′-carotenal?>?β-apo-4′-carotenal?>?γ-carotene. The enzyme converted these carotenoids to β-apo-13-carotenones by cleaving their C₁₃–C₁₄ double bonds. The oxygen atom of β-apo-13-carotenone originated not from water but from molecular oxygen. Thus, the enzyme was an apo-carotenoid 13,14-dioxygenase.     

Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation

The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M_r 23 kDa) and 218 amino acids (β subunit, M_r 24 kDa) and the NHase activator of 413 amino acids (M_r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.     

Cold adapted features of Vibrio salmonicida catalase: characterisation and comparison to the mesophilic counterpart from Proteus mirabilis

The gene encoding catalase from the psychrophilic marine bacterium Vibrio salmonicida LFI1238 was identified, cloned and expressed in the catalase-deficient Escherichia coli UM2. Recombinant catalase from V. salmonicida (VSC) was purified to apparent homogeneity as a tetramer with a molecular mass of 235 kDa. VSC contained 67% heme b and 25% protoporphyrin IX. VSC was able to bind NADPH, react with cyanide and form compounds I and II as other monofunctional small subunit heme catalases. Amino acid sequence alignment of VSC and catalase from the mesophilic Proteus mirabilis (PMC) revealed 71% identity. As for cold adapted enzymes in general, VSC possessed a lower temperature optimum and higher catalytic efficiency (k _cat/K _m) compared to PMC. VSC have higher affinity for hydrogen peroxide (apparent K _m) at all temperatures. For VSC the turnover rate (k _cat) is slightly lower while the catalytic efficiency is slightly higher compared to PMC over the temperature range measured, except at 4°C. Moreover, the catalytic efficiency of VSC and PMC is almost temperature independent, except at 4°C where PMC has a twofold lower efficiency compared to VSC. This may indicate that VSC has evolved to maintain a high efficiency at low temperatures.     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.     

Immobilized papain on gold nanorods as heterogeneous biocatalysts

Papain, a thiol protease present in the latex of Carica papaya, is an enzyme which exhibits broad proteolytic activity, and, for this reason, it is utilized in a variety of industrial applications. Immobilization of papain on gold nanoparticles highly preserves its activity and enhances the stability, allowing the reuse of the linked enzyme many times without any significant loss of its catalytic performance. In particular, k _cat and K _M values remain substantially unchanged, while immobilized form shows a higher activity on a wider pH range retains 80 % residual activity also at 90 °C and shows higher functionality than the free form when incubated for long time (1 h) at 90 °C and at extreme pH values (3 and 12). A higher activity of immobilized papain with respect to the free form in the presence of various bivalent metal ions, known as strong inhibitors of papain, was also found. The reasons of this enhanced stability of gold nanorods immobilized papain are discussed.     

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium <Emphasis Type="Italic">Alteromonas</Emphasis> sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni²⁺ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a K_m of 0.55 mM, a V_max of 83.6 U/mg, a k_cat of 74.3 s^-1 and k_cat/K_m of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.     

Purification and characterisation of lignin peroxidase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K _m, k _cat and k _cat/K _m values of the enzyme using veratryl alcohol and H₂O₂ as the substrate were 61 M, 2.13 s⁻¹, 3.5 × 10⁴ M⁻¹s⁻¹ and 71 M, 2.13 s⁻¹, 3.0 × 10⁴ M⁻¹ s⁻¹ respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

An alkali tolerant α-l-rhamnosidase from Fusarium moniliforme MTCC-2088 used in de-rhamnosylation of natural glycosides

Analkali tolerant α-l-rhamnosidase has been purified to homogeneity from the culture filtrate of a new fungal strain, Fusarium moniliforme MTCC-2088, using concentration by ultrafiltration and cation exchange chromatography on CM cellulose column. The molecular mass of the purified enzyme has been found to be 36.0 kDa using SDS-PAGE analysis. The K_m value using p-nitrophenyl-α-l-rhamnopyranoside as the variable substrate in 0.2 M sodium phosphate buffer pH10.5 at50 °C was 0.50 mM. The catalytic rate constant was15.6 s⁻¹giving the values of k_cat/K_m is 3.12 × 10⁴M⁻¹ s⁻¹. The pH and temperature optima of the enzyme were 10.5 and 50 °C, respectively. The purified enzyme had better stability at 10 °C in basic pH medium. The enzyme derhamnosylated natural glycosides like naringin to prunin, rutin to isoquercitrin and hesperidin to hesperetin glucoside. The purified α-l-rhamnosidase has potential for enhancement of wine aroma.     

Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library

There is a great interest in xylanases due to the wide variety of industrial applications for these enzymes. We cloned a xylanase gene (xyn8) from an environmental genomic DNA library. The encoded enzyme was predicted to be 399 amino acids with a molecular weight of 45.9 kD. The enzyme was categorized as a glycosyl hydrolase family 8 member based on sequence analysis of the putative catalytic domain. The purified enzyme was thermolabile, had an activity temperature optimum of 20°C on native xylan substrate, and retained significant activity at lower temperatures. At 4°C, the apparent K _m was 3.7 mg/ml, and the apparent k _cat was 123/s.Reference to a company and/or products is only for the purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Discovery and characterization of a putrescine oxidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> NCIMB 11540

A gene encoding a putrescine oxidase (PuO_Rh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K _M = 8.2 μM, k _cat = 26 s⁻¹). PuO_Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO_Rh is a reasonably thermostable enzyme with t _1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO_Rh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO_Rh. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties

Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l⁻¹) were produced in response to Cu²⁺. Highest laccase levels (19,000 U l⁻¹) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M _w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C, and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4°C. Kinetic constants (K _m, k _cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

Cloning, Expression, and Enzyme Characterization of an Acid Heat-Stable Phytase from Aspergillus fumigatus WY-2

A novel thermostable phytase gene was cloned from Aspergillus fumigatus WY-2. It was 1459 bp in size and encoded a polypeptide of 465 amino acids. The gene was expressed in Pichia pastoris GS115 as an extracellular enzyme. The expressed enzyme was purified to homogeneity and biochemically characterized. The purified enzyme had a specific activity of 51 U/mg with an approximate molecular mass of 88 kDa. The optimum pH and temperature for activity were pH 5.5 and 55°C, respectively. After incubation at 90°C for 15 min, it still remained at 43.7% of the initial activity. The enzyme showed higher affinity for sodium phytate than other phosphate conjugates, and the K_m and K_cat for sodium phytate were 114 μM and 102 s⁻¹, respectively. Incubated with pepsin at 37°C for 2 h at the ratio (pepsin/phytase, wt/wt) of 0.1, it still retained 90.1% residual activity. These exceptional properties give the newly cloned enzyme good potential in animal feed applications.     

Screening,partial purification and characterization of the hyper-thermophilic lipase produced by a new isolate of Bacillus subtilis LP2

Abstract

Lipases are one of the most important catalysts for several industries such as detergent, dairy, and textile industry due to their bio-catalytic ability in aqueous and non-aqueous media. Stability to extreme conditions is an important property since it makes enzymes suitable to several industrial processes. In this study, lipase producing soil bacteria were screened and identified with 16S rDNA sequencing. A new hyper-thermophilic lipase named as Bacillus subtilis LP2 isolate was partially purified by ammonium sulphate precipitation with 17.8-fold purification and 583?U/mg specific activity. Maximum activity was exhibited at pH 7 and 80?°C with the substrate tween 80?K_M and V_max values were calculated as 18.3?mM and 680?U/mg with a catalytic efficiency (k_cat/K_M) of 307?s^?1M^?1. These results indicate that lipase from Bacillus subtilis LP2 can be a valuable candidate for industrial applications such as organic synthesis and fats and oils industry due to their efficient catalysis in higher temperatures.     

PURIFICATION AND PHYSICOKINETIC CHARACTERIZATION OF A GLUCONOLACTONE INHIBITION-INSENSITIVE β-GLUCOSIDASE FROM Andrographis paniculata NEES. LEAF

A gluconolactone inhibition-insensitive β-glucosidase from Andrographis paniculata (Acanthaceae) leaves has been isolated, homogeneity purified, and characterized for its physicokinetic properties. The purified enzyme appeared to be a monomeric structure with native molecular weight about 60 kD. The enzyme exhibited optimum pH 5.5 and pI 4.0, meso-thermostability and high temperature optimum (55°C) for catalytic activity, with activation energy of 6.8 kcal Mol^?1. The substrate saturation kinetics studies of the enzyme revealed a Michaelis–Menten constant (K_m) of 0.25 mM for pNPG and catalytic efficiency (K_cat/K_m) of 52,400 M ^?1 s^?1, respectively. Substrate specificity of the enzyme was restricted to β-linked gluco-, manno- and fuco-conjugates. The gluconolactone inhibition insensitivity was evident from its very low inhibition at millimolar inhibitor concentrations. Interestingly, the enzyme showed geraniol transglucosylating activity with pNPG as glucosyl donor but not with cellobiose. The catalytic activity of the enzyme has been reported to be novel with respect to its activity and preferences from a medicinal plant resource.