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.     

A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

A new fungal peroxidase with alkaline-tolerant,chloride-enhancing activity and dye decolorization capacity

A new fungal peroxidase (Pspd) from Perenniporia subacida was purified by ammonium sulfate precipitation, DEAE-cellulose DE52 anionic exchange and Sepharose GL-6B chromatography, resulting in a high specific activity of 9.138 U mg⁻¹, 3.622-fold higher than that of crude enzyme at the same level. Polyacrylamide gel electrophoresis and UV–vis adsorption spectrum analysis showed that the purified enzyme is a heme-containing monomer with a molecular mass of 43.0 kDa. Optimal peroxidase activity was obtained at pH 5.5 and 30 °C when using 100.0 mM n-propanol as substrate, and under these conditions, the catalytic efficiency (k_cat/K_m) is 1.57 s⁻¹ μM⁻¹. Pspd was inhibited by l-cysteine, dithiothreitol, EDTA and sodium azide, but stimulated by Mn²⁺, Na⁺, Mg²⁺ and K⁺. The enzyme is stable over a broad pH range of 7.0–8.5 after incubation for 72 h, which indicated that the enzyme is lasting alkaline-tolerant. It was worth noting that the chloride at relatively low concentrations can enhance the peroxidase activity, with concomitant increase in substrate affinity. Additionally, Pspd performed high decolorization capability toward structurally various dyes and the capability was independent of the oxidizing mediators, with 75.31% of Neutral Red (50.0 mg L⁻¹) being decolorized by 1.5 U mL⁻¹ pure enzyme after incubation for 72 h. These properties demonstrated that Pspd has potentials for textile dyes decolorization applications.     

Purification and characterization of a nitroreductase from the soil bacterium Streptomyces mirabilis

A NADH-dependent nitroreductase from an efficient nitro-reducing soil bacterium, Streptomyces mirabilis DUT001, was isolated and characterized. The enzyme was purified to near homogeneity using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography. The native enzyme was estimated by gel filtration to have a molecular weight of 68 kDa, and its subunit molecular weight determined by SDS-PAGE was about 34 kDa, which indicated this enzyme was a dimer. Polycyclic nitroaromatic compounds were preferred substrates for this enzyme. The purified enzyme exhibited maximum activity at pH 7.5 and 40 °C. The addition of various chemicals such as reducing agents, metal ions, and chelating agents, had effects on enzyme activity. Mg²⁺, Ca²⁺, Sr²⁺, and 1% (w/v) Triton X-100 increased activity. However, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and SDS reduced activity. The maximum reaction rate (V_max) was 64 μM min^?1 mg^?1 enzyme and the apparent Michaelis–Menten constants (K_m) for 4-nitro-1,8-naphthalic anhydride and NADH were 276 and 29 μM, respectively. Menadione, bimethylenebis, sodium benzoate, and antimycin A were inhibitors of the purified nitroreductase with apparent inhibition constants (K_is) of 20, 36, 44 and 80 μM, respectively.     

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 and characterization of a highly selective glycyrrhizin-hydrolyzing β-glucuronidase from Penicillium purpurogenum Li-3

A novel β-glucuronidase from filamentous fungus Penicillium purpurogenum Li-3 was purified to electrophoretic homogeneity by ultrafiltration, ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration with an 80.7-fold increase in specific activity. The purified β-glucuronidase is a dimeric protein with an apparent molecular mass of 69.72 kDa (m/z = 69,717), determined by MALDI/TOF-MS. The optimal temperature and pH of the purified enzyme are 40 °C and 6.0, respectively. The enzyme is stable within pH 5.0–8.0, and the temperature up to 45 °C. Mg²⁺ ions enhanced the activity of the enzyme, Ca²⁺ and Al³⁺ showed no effect, while Mn²⁺, Zn²⁺, Hg²⁺ and Cu²⁺ substantially inhibited the enzymatic activity. The K_m and V_max values of the purified enzyme for glycyrrhizin (GL) were evaluated as 0.33 mM and 59.0 mmol mg^?1 min^?1, respectively. The purified enzyme displayed a highly selective glycyrrhizin-hydrolyzing property and converted GL directly to glycyrrhetic acid mono-glucuronide (GAMG), without producing byproduct glycyrrhetic acid (GA). The results suggest that the purified enzyme may have potential applications in bio-pharmaceutical and biotechnological industry.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Microbial processes and bacterial populations associated to anaerobic treatment of sulfate-rich wastewater

A pilot-scale (1.2 m³) anaerobic sequencing batch biofilm reactor (ASBBR) containing mineral coal for biomass attachment was fed with sulfate-rich wastewater at increasing sulfate concentrations. Ethanol was used as the main organic source. Tested COD/sulfate ratios were of 1.8 and 1.5 for sulfate loading rates of 0.65–1.90 kgSO₄²⁻/cycle (48 h-cycle) or of 1.0 in the trial with 3.0 gSO₄²⁻ l⁻¹. Sulfate removal efficiencies observed in all trials were as high as 99%. Molecular inventories indicated a shift on the microbial composition and a decrease on species diversity with the increase of sulfate concentration. Beta-proteobacteria species affiliated with Aminomonas spp. and Thermanaerovibrio spp. predominated at 1.0 gSO₄²⁻ l⁻¹. At higher sulfate concentrations the predominant bacterial group was Delta-proteobacteria mainly Desulfovibrio spp. and Desulfomicrobium spp. at 2.0 gSO₄²⁻ l⁻¹, whereas Desulfurella spp. and Coprothermobacter spp. predominated at 3.0 gSO₄²⁻ l⁻¹. These organisms have been commonly associated with sulfate reduction producing acetate, sulfide and sulfur. Methanogenic archaea (Methanosaeta spp.) was found at 1.0 and 2.0 gSO₄²⁻ l⁻¹. Additionally, a simplified mathematical model was used to infer on metabolic pathways of the biomass involved in sulfate reduction.     

Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation

A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg²⁺, Zn²⁺, K⁺ and NH₄⁺. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent K_m 1.3 mg ml^?1, V_max 5.523 × 10^?5 moles l^?1 min^?1 and K_cat 2.37 s^?1 and catalytic efficiency 1.82 s^?1 M^?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 5.83 mM). Among metal ions and reagents tested, Ca²⁺ and K⁺ enhanced the enzymatic activity, but Mg²⁺, Mn²⁺, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag⁺, Hg²⁺ and Co²⁺ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose.     

Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation

The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL⁻¹) and ultrafiltration, followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 °C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba²⁺ increased the enzyme activity by 96% and its thermal stability by 30%, besides increasing its stability at acid pH. The apparent K_m with apple pectin as substrate was 1.82 mg mL⁻¹ and the V_max was 81 μmol min⁻¹ mg⁻¹.     

Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: Its expression and characterization for CO2-sequestration applications

The codon-optimized carbonic anhydrase gene of Persephonella marina EX-H1 (PMCA) was expressed and characterized. The gene with the signal peptide removed, PMCA(sp−), resulted in the production of approximately five times more purified protein than from the intact gene PMCA using an Escherichia coli expression system. PMCA(sp−) is formed as homo-dimer complex. PMCA(sp−) has a wide pH tolerance (optimum pH 7.5) and a high thermostability even at 100 °C (88 min of thermal deactivation half-life). The melting temperature for PMCA(sp−) was 84.5 °C. The apparent k_cat and K_m values for CO₂ hydration were 3.2 × 10⁵ s⁻¹ and 10.8 mM. The activity of the PMCA(sp−) enzyme was enhanced by Zn²⁺, Co²⁺, and Mg²⁺, but was strongly inhibited by Cu²⁺, Fe³⁺, Al³⁺, Pb²⁺, Ag⁺, and Hg²⁺. PMCA(sp−) readily catalyzed the hydration of CO₂, precipitating CaCO₃ as calcite in the presence of Ca²⁺.     

Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes

A yellow laccase from the culture filtrate of Trametes hirsuta MTCC-1171 has been purified. The purification methods involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 55.0 kDa. Using 2,6-dimethoxyphenol, 2,2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as the substrates, the K_m, k_cat and k_cat/K_m values of the laccase were found to be 420 μM, 13.04 s⁻¹, 3.11 × 10⁴ M⁻¹ s⁻¹, 225 μM, 13.03 s⁻¹, 1.3 × 10⁵ M⁻¹ s⁻¹ and 100 μM, 13.04 s⁻¹, 5.8 × 10⁴ M⁻¹ s⁻¹, respectively. The pH and temperature optima were 4.5 and 60 °C, respectively while pH and temperature stabilities were pH 4.5 and 50 °C. The activation energy for thermal denaturation of the enzyme was 18.6 kJ/mol/K. The purified laccase has yellow colour and does not show absorption band around 610 nm like blue laccases. The purified laccase transforms toluene, 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator molecules in high yields.     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

Purification and characterization of aminopeptidase B from goat brain

Aminopeptidase B was purified from goat brain with a purification fold of ～280 and a yield of 2.7%. The enzyme revealed a single band on both native acrylamide gel and SDS-PAGE thereby confirming apparent homogeneous preparation and its monomeric nature. The enzyme exhibited a molecular mass of 80.2 kDa and 79.7 kDa on Sephadex G-200 and SDS-PAGE respectively. The pH optimum was 7.4 and the enzyme was stable between pH 6.0 and 9.0. l-Arg-βNA was the most rapidly hydrolyzed substrate followed by Lys-βNA. The K_m value with Arg-βNA was found to be 0.1 mM. Metal chelating and –SH reactive agents strongly inhibited the enzyme activity. 1,10-Phenanthroline exhibited mixed type of inhibition with a K_i of 5 × 10^?5 M. The enzyme was highly sensitive to urea. Metal ions like Ni²⁺, Cd²⁺, Fe²⁺and Hg²⁺ inhibited the enzyme, whereas Co²⁺, Zn²⁺, Mn²⁺and Sn²⁺ slightly activated the enzyme.     

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn²⁺, K⁺, Zn²⁺, Ca²⁺ and Al³⁺ inhibited the enzyme activity; it increased in the presence of Mg²⁺ and Cu²⁺ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K_m and V_max values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T₆₅₀ from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.     

Inhibition kinetics of a commercial mixed culture by ammonium sulfate

The inhibitory effect of ammonium sulfate on a commercial mixed culture, used in biological waste-water treatment was studied under aerobic batch conditions. Several mathematical models of enzyme and growth kinetics including a death factor were analyzed through nonlinear regression to find the best fit to corresponding data of inhibition. The best fit model was found to be the generalized Monod type with a death factor having the biokinetic parameters; μ_max 0.681 h⁻¹, K_s 0.224 g dm⁻³, K_i 56240 g dm⁻³, K 0.055 g dm⁻³ and k_d 0.052 h⁻¹ to represent the experimental data accurately. The low saturation coefficient value along with high maximum specific growth rate and inhibition coefficient denotes the competitive characteristics of commercial mixed cultures in the biological treatment of high ammonium polluted waste waters.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.