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.     

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent K_m and V_max for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.     

Purification and characterization of two agarases from Agarivorans albus OAY02

Two extracellular β-agarases were purified from Agarivorans albus OAY2, which was isolated from seaweed collected in Qingdao, Shandong, China. The fermentation process was optimized with enzyme activity improving from 1.06 U/ml to 2.65 U/ml. Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in a 107-fold increase in agarase-a activity and a 52-fold increase in case of agarase-b, with high specific activities of 2715 and 1338 U/mg, respectively. The molecular masses of the agarases were estimated to be 50 kDa and 107 kDa, respectively. The optimum temperatures for the activities of agarase-a and agarase-b were 40 °C and 50 °C, respectively. Agarase-a was stable at 30 °C, while agarase-b was stable at 50 °C. The optimum pH values for agarase-a and agarase-b were both 9.0. Agarase-a was inhibited by Cu²⁺, Mn²⁺, Co²⁺ and EDTA, while agarase-b was inhibited by Cu²⁺, Mn²⁺ and Fe²⁺. The results of ¹³C NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) and the enzymatic product results indicated that agarase-a and agarase-b might be novel agarases.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Calcium dependent,alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.     

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.     

Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

Purification of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity

The fungus Scopulariopsis brevicaulis was isolated from poultry farm soil at Namakkal, India. The extracellular keratinase from this fungus was purified to homogeneity by ammonium sulphate precipitation and procedure involving DEAE-Cellulose and Sephadex G-100 chromatographic techniques. The purified enzyme was formed from a monomeric protein with molecular masses of 39 and 36 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 8.0 and the optimum temperature at pH 8.0 was 40 °C. The activity of purified keratinase with respect to pH, temperature and salt concentration was optimized by Box–Behnken design experiment. It was shown that a second-order polynominal regression model could properly interpret the experimental data with an R²-value of 0.9957 and an F-value of 178.32, based on the maximum enzyme activity examined. Calculated optimum conditions were predicted to confer a 100% yield of keratinase activity with 5 mM CaCl₂, pH 8.0 and at a temperature of 40 °C. The enzyme was strongly inhibited by PMSF, which suggests a serine residue at or near an active site. The purified keratinase was examined with its potential for dehairing the skin.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

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.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus

Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61 kDa protein with activity of 24.3 U/mg and the OD₆₀₀ of 23 was reached after 18 h induction with 10 mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50 U/ml SSbgly and 90 mg/ml Ste at 75 °C as determined by the response surface method.     

Biochemical characterization of a lichenase from Penicillium occitanis Pol6 and its potential application in the brewing industry

The purification and characterization of an extracellular lichenase from the fungus Penicillium occitanis Pol6 were studied. The strain produced the maximum level of extracellular lichenase (45 ± 5 U ml⁻¹) when grown in a medium containing oat flour (2%, w/v) at 30 °C for 7 days. The purified enzyme EG_L showed as a single protein band on SDS–PAGE with a molecular mass of 20 kDa. Its N-terminal sequence of 10 amino acid residues was determined as LDNGAPLLNV. The purified enzyme showed an optimum activity at pH 3.0 and 50–60 °C. The half-lives of EG_L at 60 °C and 70 °C were 80 min and 21 min, respectively. Substrate specificity studies revealed that the enzyme is a true β-1,3-1,4-d-glucanase. The enzyme hydrolyzed lichenan to yield trisaccharide, and tetrasaccharide as the main products. Under simulated mashing conditions, addition of EG_L (20 U/ml) or a commercial β-glucanase (20 U/ml) reduced the filtration time (25% and 21.3%, respectively) and viscosity (10% and 8.18%, respectively). These characteristics indicate that EG_L is a good candidate in the malting and brewing industry.     

Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations

An extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4) was purified from the optimized culture medium. The SAL4 specific activity determined at 60 °C and pH 12 by using olive oil emulsion or TC4, reached 7215 U/mg and 2484 U/mg, respectively. The 38 NH₂-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK) was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0–12 after a 24 h incubation or after 0.5 h treatment at 70 °C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

A novel thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production

A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5–11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12 h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10 mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24 h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2–6.     

Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability

A putative laccase gene was cloned from Shigella dysenteriae W202 and expressed in Escherichia coli as a soluble fusion protein with high yield. The purified product (Wlac) was characterized as the CueO-like laccase from E. coli, a monomer of molecular mass 55 kDa, with a maximum activity of 24.4 U/mg (K_m = 0.086) and a pH optimum of 2.5, in a standard assay using ABTS (2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonate) as the substrate. Activity was stable at 0–25 °C but inhibited above 40 °C. Purified Wlac was completely inhibited by 200 mM EDTA and partially by 32 mM SDS, 50 mM NaN₃ and 60 mM thioglycolic acid. Activity was stimulated by Cu²⁺; other metal ions had only slight or negative effects. Two mutated variants, WlacS and WlacD, were obtained by substituting Glu 106 with Phe 106, and adding a deletion of an α-helix domain (from Leu 351 to Gly 378). WlacS had a 2.2-fold (52.9 U/mg) and WlacD a 3.5-fold (85.1 U/mg) higher enzyme activity than the wild-type laccase and WlacD showed greater thermostability at higher temperatures. Sce VMA intein-associated fusion proteins maintained ～80% of total enzyme activity. Thus, deletion and site-directed mutagenesis of laccases are capable of promoting both enzymatic activity and thermostability.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.     

Characterization,purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica)

In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l-tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l-cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of E_a 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.     

Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.