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.     

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.     

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.     

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.     

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.     

Immobilization of glycolate oxidase from Medicago falcata on magnetic nanoparticles for application in biosynthesis of glyoxylic acid

Glycolate oxidase was isolated from Medicago falcata Linn. after a screening from 13 kinds of C₃ plant leaves, with higher specific activity than the enzyme from spinach. The M. falcata glycolate oxidase (MFGO) was partially purified and then immobilized onto hydrothermally synthesized magnetic nanoparticles via physical adsorption. The magnetic nanoparticles were characterized with scanning electron microscope (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. The maximum load of MFGO was 56 mg/g support and the activity recovery was 45%. Immobilization of MFGO onto magnetic nanoparticles enhanced the enzyme stability, and the optimum temperature was significantly increased from 15 °C to 30 °C. The immobilized biocatalyst was successfully used in a batch reactor for repeated oxidization of glycolic acid to synthesize glyoxylic acid, retaining ca. 70% of its initial activity after 4 cycles of reaction at 30 °C for nearly 70 h, and its half-life was calculated to be 117 h.     

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.     

A novel low-temperature-active exo-inulinase identified based on Molecular-Activity strategy from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis

A novel glycosyl hydrolase family 32 exo-inulinase (InuAGN25) gene was cloned from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis. InuAGN25 showed the highest identity of 54.3% with a putative levanase recorded in GenBank. Molecular-Activity strategy was proposed to predict InuAGN25 to be a low-temperature-active exo-inulinase before experiments performance. Molecular analyses included progressive sequential, phylogenetic and structural analyses. InuAGN25 was effectively expressed in Escherichia coli. The purified recombinant InuAGN25 showed characteristics of low-temperature-active enzymes: (1) the enzyme retained 55.8% of the maximum activity at 20 °C, 35.8% at 10 °C, and even 8.2% at 0 °C; (2) the enzyme exhibited 75.8, 30.5 and 10.8% of the initial activity after preincubation for 60 min at 45, 50 and 55 °C, respectively; (3) K_m values of the enzyme toward inulin were 2.8, 3.0, 3.2 and 5.8 mg ml⁻¹ at 0, 10, 20 and 40 °C, respectively. Fructose was the main product of inulin and Jerusalem artichoke tubers hydrolyzed by the purified recombinant InuAGN25 at room temperature, 10 °C and 0 °C. These results suggested the Molecular-Activity strategy worked efficiently and made InuAGN25 promising for the production of fructose at low temperatures.     

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ～456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T_1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol^?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg²⁺ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) for sodium phytate were 83 nmol mg^?1 s^?1 and 0.156 mM, respectively. The catalytic turnover number (K_cat) and catalytic efficiency (K_cat/K_m) of phytase were 37.8 s^?1 and 2.4 × 10⁵ M^?1 s^?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.     

Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).     

Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils

Lipase production (8.02 ± 0.24 U/ml) by the yeast Aureobasidium pullulans HN2.3 isolated from sea saltern was carried by using time-dependent induction strategy. The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 °C, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.     

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

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

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.     

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.     

Fructose-1,6-bisphosphatase from a hyper-thermophilic bacterium Thermotoga maritima: Characterization,metabolite stability,and its implications

Fructose-1,6-bisphosphatase gene from a hyperthermophilic bacterium Thermotoga maritima was cloned, and the recombinant protein was produced in E. coli, purified, and characterized. The fructose-1,6-bisphosphatase (FBPase) with a molecular mass of ca. 28 kDa was purified from the fusion protein cellulose-binding module (CBM)-intein-FBPase by affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. The substrate fructose 1,6-bisphosphate was not stable at high temperatures, especially at high pHs. The degradation constants of fructose 1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate were determined at different temperatures (37, 60, and 80 °C) and pH 7.5 or 9.0. The k_cat and K_m values of FBPase were 8.57 s⁻¹ and 0.04 mM at 60 °C, as well as 58.7 s⁻¹ and 0.12 mM at 80 °C. This enzyme was very stable at its suboptimal temperatures, with half-life times of ca. 1330 and 55.6 h at 60 and 80 °C, respectively. At 60 °C, this enzyme had an estimated total turn-over number of 20,500,000 (mol product/mol enzyme) and weight-based total turn-over umber of 192,000 (kg product/kg enzyme), respectively. These results indicated that this enzyme would be a stable building block for cell-free synthetic pathway biotransformation (SyPaB) that can implement complicated biochemical reactions. In order to obtain high-yield desired products, we suggest that over-addition or over-expression of the enzymes responsible for converting easily degraded metabolites should be important to prevent unnecessary metabolite loss for in vitro or in vivo synthetic pathway design.     

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45 kDa, 55 °C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138 U mL^?1 at 44 h, and the productivity reached 48.58 U (mL h)^?1 through the homologous overexpression of strain B-pN-pelA in a 7.5 L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50 °C for 4 h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.     

Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization

Two thermostable and alkali-stable β-1,3–1,4 glucanases (EC 3.2.1.73) EG1 and EG2 from a newly isolated Bacillus licheniformis UEB CF were purified. The molecular weights of EG1 and EG2 enzymes determined by SDS–PAGE were approximately 30 kDa and 55 kDa, respectively. The N-terminal amino acid sequences of EG1 and EG2 β-glucanases were determined to be GAAPIKKGTTKLN and DINGGGATLPQK, respectively. The optimum temperature, optimum pH, k_m and V_max of EG1 were 70 °C, 5.0, 2.1 mg/ml and 21.25 μmol/min/mg, respectively. These values for EG2 were 60 °C, 7.0, 1.82 mg/ml and 18.54 μmol/min/mg, respectively.Both endoglucanases were highly active against barley β-glucan and lichenan. However, they were inactive against CMC and laminarin. The purified β-glucanases were found to be relatively stable toward non-ionic surfactants and oxidizing agents. In addition, both enzymes showed excellent stability and compatibility with a wide range of commercial solid detergents suggesting that they are a potential candidate in detergent industries formulation.     

Purification and partial characterisation of a thermostable xylanase from salt-tolerant Thermobifida halotolerans YIM 90462T

ThxynA, an extracellular xylanase of T. halotolerans YIM 90462^T, was purified to homogeneity from a fermentation broth by ultra-filtration, ammonium sulphate precipitation, hydrophobic chromatography and ion exchange chromatography. The purified xylanase has a molecular mass of 24 kDa and is optimally active at 80 °C and pH 6.0. The enzyme is stable over a broad pH range (pH 6.0–10.0) and shows good thermal stability when incubated at 70 °C for 1 h. The K_m and V_max values of the enzyme are 11.6 mg/mL and 434 μmol mg^?1 min^?1, respectively, using oat spelt xylan as a substrate. Moreover, the enzyme seemingly has both xylanase activity and cellulase activity. These unique properties suggest that it may be useful for industrial applications.     

Application of statistical experimental design for optimization of keratinases production by Bacillus pumilus A1 grown on chicken feather and some biochemical properties

A new keratinolytic enzyme-producing bacterium was isolated from slaughter house polluted water and identified as Bacillus pumilus A1. Medium composition and culture conditions for the keratinases production by B. pumilus A1 were optimized using two statistical methods: Plackett–Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and central composite design used to optimize the concentration of the five significant variables: feathers meal, soy peptone, NaCl, KCl, and KH₂PO₄. The medium optimization resulted in a 3.4-fold increase in keratinase production (87.73 U/ml) compared to that of the initial medium (25.9 U/ml). The zymography analysis shows the presence of at least five keratinolytic enzymes. The keratinolytic activity of the extracellular proteinases was examined by incubation with non-autoclaved chicken feathers. Complete solubilisation of whole feathers was observed after a 6-h incubation at temperatures ranging from 45 °C to 60 °C. The crude enzyme exhibited maximal activity at 60 °C and pH 8.5 or 55 °C and pH 9.0 using casein or keratin as substrates, 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.