Characterisation of acid proteases from a fusant F76 and its progenitors Aspergillus oryzae HN3042 and Aspergillus niger CICC2377

The characteristics of a novel acid protease from a fusant F76 were comparatively evaluated with those from its progenitors Aspergillus oryzae HN3042 and A. niger CICC2377. The UV spectra of these three acid proteases were similar, but fluorescence spectra were different. The acid protease from F76 contained 7.1% α‐helix, 39.4% β‐sheet, 24.7% β‐turn and 32% aperiodic coil, unlike those from its progenitors. The acid protease from F76 was active in the temperature range of 35–55 °C with the optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with the optimum pH of 3.5, while those values from A. oryzae HN3042 and A. niger CICC2377 were 45 °C, 4.0 and 40 °C, 3.5, respectively. The kinetic parameters of the acid protease from F76 were different from its progenitors and the Michaelis constant, maximum velocity, activation energy, and attenuation index were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1 and 0.59, respectively.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillus borstelensis (P35) and its applications in fruit juice and oil extraction

An alkaline pectin lyase (PNL) (EC 4.2.2.10) secreted by Brevibacillus borstelensis P35 (GenBank Number: FJ417406) was purified using ammonium sulfate fractionation, anion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-150. The pH and temperature optima of the enzyme were found to be 8.0 and 60 °C. The enzyme does not loose activity up to 60 °C if exposed for 1 h. The values of K _m and V _max of the enzyme were 0.625 mg/mL and 126.32 s^?1, respectively. The molecular weight was found to be 36 ± 01 kDa. The presence of 10 mM concentration of Ca²⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Hg²⁺, Fe²⁺ and EDTA, l-cystein, ascorbic acid significantly enhanced the PNL of the purified enzyme. In the course of the laboratory trials, it was demonstrated that PNL from B. borstelensis (P35) could be successfully applied to the production and clarification of fruit juice and oil extraction.     

Purification and characterization of an alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 and its application in mannooligosaccharides preparation having prebiotic potential

An alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 was purified 38.96-fold to homogeneity with specific activity of 2,288.90 ± 27.80 U mg^?1 protein and final recovery of 8.92 ± 0.09 %. The purified β-mannanase was an extracellular monomeric protein with a molecular mass of 50 kDa on SDS–PAGE. The first 20 N-terminal amino acid sequence of mannanase enzyme was MVVKKLSSFILILLLVTSAL. The optimal temperature and pH for enzyme were 65 °C and 8.8, respectively. It was completely stable at 60 °C for 3 h and retained >50 ± 1.0 % activity at 70 °C up to 3 h. The β-mannanase was highly stable between pH 5–10 and retained >85 % of the initial activity for 3 h. The metal ions Ni⁺², Co⁺², Zn⁺² and Mg⁺² enhanced the enzyme activity. The enzyme remained stable after 3 h of preincubation with most of the tested organic solvents. According to substrate specificity study, the purified mannanase had high specificity to locust bean gum which was degraded mainly to mannooligosaccharides (MOS) like mannotriose, mannotetraose and mannopentose. These MOS enhanced the growth of Lactobacillus casei but inhibited the growth of Salmonella enterica indicating potential prebiotic properties. The properties of the purified β-mannanase from B. nealsonii PN-11 make this enzyme attractive for biotechnological applications.     

Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a

The aim of this study was to purify a malolactic enzyme (MLE) from Oenococcus oeni (O. oeni) strain and determine its properties in detail. O. oeni SD-2a was cultivated in the ATB broth supplemented with 7 g/L l-malic acid for harvesting the cells. After harvest, the cells were washed and disrupted for purification of MLE. MLE was purified from the supernatant of the disrupted cells through protamine sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purified MLE was identified using mass spectrometry. The MLE was purified by 43-fold with a yield of 0.42 % and possessed a specific activity of 419.2 U/mg. The purified enzyme with a nominal molecular mass of 59 kDa and a theoretical pI of 4.76 exhibited a maximum enzyme activity at 35 °C and pH 6.0, which retained over 50 % of its initial activity in the presence of 14 % (v/v) ethanol. Mn²⁺ was proven to be the most effective divalent cation to promote enzyme activity. Under the conditions of temperature 30 °C and pH 6.0, the K _m and V _max of MLE on l-malic acid were 12.5 × 10^?3 M and 43.86 μmol/(min × mg), respectively. Moreover, the purified enzyme exhibited a higher stability with 0.1 M NaCl in addition and had a half-life of 30 days at 4 °C.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The K_m and V_max values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni²⁺, Al³⁺, and Fe²⁺ and was increased in the presence of Ca²⁺ and Mg²⁺ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.     

Characterization of amylase and protease produced by Aspergillus awamori in a single bioreactor

The aim of this study was to characterize the glucoamylase and acid protease produced in a single bioreactor by Aspergillus awamori: nakazawa MTCC 6652. Both the enzymes were found stable in wide range of pH (3–9) and temperature (25–70 °C). Optimum activities of amylase and protease were obtained at pH 4 and 5, respectively, whereas 70 and 55 °C had been found as most suitable temperature for highest activities of amylase and protease, respectively. Half life of glucoamylase was 210, 120, 60 and 35 min at 50, 60, 70 and 80 °C, respectively, which was 150, 120, 65 and 15 min at 40, 50, 60 and 70 °C, respectively, for acid protease. K_m and V_max of glucoamylase and protease were 9.8 mg/ml, 56.2 mg/ml/min and 1.08 mg/ml, 8.8 mg/ml/min, respectively. In low amount (1 mM) almost all metal ions except Mn, such as Ca²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, Zn²⁺ and Hg²⁺ enhanced glucoamylase activity whereas protease activity was inhibited by all the ions except Zn²⁺. At low concentration, i.e., (0.03% w/v) Triton X-100 and SLS increased the activity of glucoamylase, while in higher concentration it inhibited activities of both the enzymes. β-mercaptoethanol at 0.25% (v/v) enhanced the amylase and protease activity by 1.6 and 3.0 fold, respectively, whereas the presence of 0.5% (v/v) β-mercaptoethanol inhibited the activities of both the enzymes drastically. At 0.5 M concentration of urea, glucoamylase activity increased but drastic inhibition took place at 5 M urea. In case of protease, 0.5 M of urea enhanced its activity and 1 M urea inhibited it completely. Thus, glucoamylase and protease produced by A. awamori nakazawa confirm their suitability for diverse applications in industries.     

Synergistic Processing of Skim Milk with High Pressure Nitrous Oxide,Heat, Nisin,and Lysozyme to Inactivate Vegetative and Spore-Forming Bacteria

Individual and combined effects of high pressure nitrous oxide (HPN₂O), heat, and antimicrobials on the inactivation of Escherichia coli, Listeria innocua, and Bacillus atrophaeus endospores in milk were all evaluated after 20-min treatments. Stand-alone milk treatments with HPN₂O (15.2 MPa), heat (45 and 65 °C), or nisin (50 and 150 IU mL^?1) resulted in log₁₀ reductions ranging only from 0.1 to 2.1 for E. coli and L. innocua. Combining HPN₂O (15.2 MPa) with heat (65 °C) inactivated 6.0 and 5.1 log₁₀ in the vegetative bacteria, respectively. Similarly, reductions of 5.9 and ≥ 6.0 log₁₀ of respective E. coli and L. innocua cells in milk were achieved through a combination of HPN₂O (15.2 MPa), heat (65 °C), and nisin (150 IU mL^?1). A 2.5 log₁₀ cycle inactivation of spores was obtained by HPN₂O, nisin (at both 50 and 150 IU mL^?1), and lysozyme (50 μg mL^?1) at 85 °C. Combining these processing techniques resulted in significantly greater microbial inactivation (p < 0.05) than the sum of individual reductions from each treatment alone, indicating synergistic effects. HPN₂O irrespective of processing temperatures did not cause any occurrence of sub-lethally injured cells or disruption in colloidal stability of milk at 65 and 85 °C (p ≥ 0.05). Color and pH changes in milk following the most demanding treatment conditions were minimal.     

Purification and characterization of lysozyme from filipino venus, Ruditapes philippinarum

Lysozyme from Filipino venus (Ruditapes philippinarum) was purified by ion-exchange and gel filtration chromatography. The purification fold and yield were 3,402 and 32.4%, respectively. The molecular weight was determined to be 13.4 kDa by SDS-PAGE. The specific activity of lysozyme was 3.76×10⁵ units/mg protein with Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of lysozyme were 75°C and 5.5, respectively. Lysozyme activity was decreased with about 45% after heat treatment for 30 min at 80°C, and completely inactivated at 100°C. It was activated by NaCl (10–70 mM), MgCl₂, and CaCl₂ (2–5 mM) whereas it was inhibited by ZnCl₂ (2–30 mM).     

Characterization of starch and cell walls from mature-green ambarella (Spondias cytherea Sonnerat) and their enzymatic hydrolysis

Juice from mature-green ambarella contains starch, a characteristic detrimental to its visual appearance due to the white sediment formed upon storage. The purpose of this work was to evaluate the effects of starch and cell wall degrading enzymes on juice residual starch and content in soluble sugars. Starch and cell walls from mature-green ambarella fruits were purified and characterized. Starch was found to contain 21.0% amylose, 78.1% amylopectin and 0.9% other minors compounds. Its average granule size was 20 μm. Its thermal characteristics were: temperatures of onset (T _o = 57.8 °C), peak (T _p = 65.6 °C), and conclusion (T _c = 72.6 °C) of gelatinization and the endothermic enthalpy (ΔH _{gelatinisation} = 12.4 J g^?1). Cell walls represented 2.8% of the edible fresh matter and were mainly constituted of highly methylated (HM) pectic substances and cellulose. The amylolytic preparations we tested, AMG^® 300 L and Hazyme^® C, showed similar behaviours in terms of starch hydrolysis and profit of Brix degree obtained. With 200 μg g^?1 of AMG^® 300 L or Hazyme^® C, the degree of amylolysis of coarse ambarella puree was close to 50% and its increased up to 70% with enzymes concentrations higher than 1,000 μg g^?1 (gelatinization at 75 ± 5 °C for 15 min followed by starch amylolysis at 60 ± 5 °C for 15 min). Total hydrolysis of ambarella starch is possible when pectinolysis occurred before amylolysis treatment, probably because of the fluidification of the medium by the pectocellulolytic enzymes. Pectinex^® Ultra SP-L was the most efficient preparation to degrade the ambarella pectins (～80% of cell wall uronides liberated from 120 mg g^?1 of purified cell walls within 1 h at 30 °C, pH 2.7).     

Ultrasonic-Assisted Extraction of α-Tocopherol Antioxidants from the Fronds of Elaeis guineensis Jacq.: Optimization, Kinetics, and Thermodynamic Studies

This article presents the first report on the extraction and quantification of α-tocopherol from the fronds of the oil palm (Elaeis guineensis Jacq). In this study, the optimization, kinetic, and thermodynamic data of α-tocopherol extraction by sonication are presented. Response surface methodology coupled with central composite design was used to optimize the experimental conditions for α-tocopherol extraction. Three independent variables, namely sample/solvent ratio (1:20–1:40 g/ml), extraction temperature (30–50 °C), and extraction time (20–50 min) were studied. For optimum conditions of 39.31 °C (~40 °C), 50 min, and 1:23.63 g/mL (~1:20 g/mL), total tocols and α-tocopherol optimal concentrations were 346.49 μg/g oil palm fronds (OPFs) by dry weight (DW) and 28.41 μg/g OPF DW, respectively. The effects of extraction temperature and tocol concentrations on the extraction kinetics and thermodynamic parameters were also studied. From the mass transfer rate equation, the kinetic and thermodynamic data obtained from the ultrasonic-assisted extraction (UAE) of α-tocopherol from OPF were activation energy, E _a (104.6 kJ mol^?1), UAE rate constant, k (6.886?×?10^?3 min^?1), ΔH (+0.818 kJ mol^?1), ΔS (+27.22 J mol^?1 K^?1), and ΔG (?8.52 J mol^?1). According to this study, the UAE of α-tocopherol from OPF is endothermic, irreversible, and spontaneous.     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

Helvellisin,a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa

A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. It was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 °C, with about 40% activity remaining at 87 °C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14 600 U/mg toward casein. The K_m of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 °C. The V_max was 5.35 × 10^− 2 mg ml^− 1 min^− 1. It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg²⁺, Fe²⁺ and Mn²⁺, but was curtailed by Cu²⁺, Hg²⁺ and Fe³⁺. It was devoid of antifungal and ribonuclease activities.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Simultaneous Detection of Salmonella,Listeria monocytogenes,and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting

A method combining multiplex real-time polymerase chain reaction (PCR) with high-resolution melting (HRM) analysis for rapid and specific simultaneous detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus was developed. The method included a melting-curve analysis of products and was evaluated by specificity, sensitivity and reproducibility analyses. Sensitivity and reproducibility analyses was both conducted by genomic DNA extracted from serial dilutions for each target pathogen. Assays with artificially inoculated and naturally contaminated samples after enrichment were also conducted. In the specificity test, there was no nonspecific amplification of the 44 nontarget pathogens, whereas the actual T _m values were 79.38?±?0.14, 82.54?±?0.15, and 77.36?±?0.14 °C for Salmonella, L. monocytogenes, and S. aureus, respectively. The sensitivity of the method was 3.5?×?10² CFU ml^?1 for Salmonella and L. monocytogenes and 3.5?×?10³ CFU ml^?1 for S. aureus. The coefficients of variation of T _m values ranged 0.51–1.03 % for Salmonella, 1.63–2.11 % for L. monocytogenes, and 0.75–2.17 % for S. aureus in intraassay, and ranged 0.81–2.43 % for Salmonella, 1.97–2.35 % for L. monocytogenes, and 0.93–3.93 % for S. aureus in interassay. The detection limit in artificially inoculated samples (n?=?50) was 5 CFU (25 g)^?1 food for the three tested pathogens. In the naturally contaminated samples (n?=?120),Salmonella DNA was detected by HRM, sequencing, and conventional culture-based methods at a positive rate of 25.00, 25.00, and 24.17 %, respectively; the corresponding rates for L. monocytogenes were 14.17, 14.17, and 14.17 %, respectively, while those for S. aureus were 16.7, 16.7, and 16.7 %, respectively.     

Constitutive expression,purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry

BACKGROUND: Pectin methylesterase (PME) catalyses the hydrolysis of the methyl ester of pectin, yielding free carboxyl groups and methanol. PME is widely used in the food, cosmetic and pharmaceutical industries. RESULTS: PME from Aspergillus niger was constitutively expressed to a high level in the yeast Pichia pastoris. The recombinant PME was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, giving an overall yield of 28.0%. It appeared as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a molecular mass of about 45 kDa. Optimal activity of the enzyme occurred at a temperature of 50 °C and a pH of 4.7. The K_m, V_max and k_cat values of the enzyme with respect to pectin were 8.6 mmol L^?1 [ ], 1.376 mmol min^?1 mg^?1 and 8.26 × 10² s^?1 respectively. Cations such as K⁺, Mg²⁺, Ni²⁺, Mn²⁺ and Co²⁺ slightly inhibited its activity, whereas Na⁺ had no effect. CONCLUSION: PME from A. niger was constitutively expressed to a high level in P. pastoris without methanol induction. The recombinant PME was purified and characterised and shown to be a good candidate for potential application in the fruit juice industry. Copyright © 2012 Society of Chemical Industry     

Kinetic studies on strawberry anthocyanin hydrolysis by a thermostable anthocyanin-β-glycosidase from Aspergillus niger

Several kinetic characteristics of a thermostable anthocyanin-β-glycosidase from Aspergillus niger have been evaluated. With strawberry anthocyanins as substrate, at pH optimum (4·0) and t = 30°C, K_m was found to be $\text{123 ± 4 μ}\text{m}$ and V_max, 1·16 ± 0·06 μmol min^?1mg^?1 protein. Temperature optimum was observed at about 68°C. The apparent energy of activation was calculated to be 11 ± 1 kcal/mol. The inhibitory effect of different sugars and sugar derivatives was examined. Glucono-deltalactone ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2·3 ± 0·1 μ}\text{m}$), gluconic acid ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 82 ± 2 μ}\text{m}$) and glucose ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1·3 ± 0·1}\text{m}\text{m}$) appeared to be competitive inhibitors of this enzyme.     

Alternative Pressing/Ultrafiltration Process for Sugar Beet Valorization: Impact of Pulsed Electric Field and Cossettes Preheating on the Qualitative Characteristics of Juices

Alternative process of sugar beet transformation is investigated by tuning experimental conditions. A three-step process has been set-up: (1) sugar beet cossettes pretreatment by pulsed electric field (PEF) and (or) short preheating to different temperatures; (2) extraction of juice from pre-treated cossettes by pressing; and (3) purification of the expressed juice by ultrafiltration. The PEF treatment was applied to cold (10 °C) and preheated (to 20, 50, 60, 70, and 80 °C) sugar beet cossettes with intensity of E?=?600 V cm^?1 using rectangular monopolar pulses of 100 μs during t _PEF?=?5–20 ms. Experiments were performed with cossettes of three sizes. Control experiments were done without PEF treatment using cold (10 °C) and preheated (to 20–80 °C) cossettes. PEF-treated and (or) preheated cossettes were pressed at 5 bars during 15 min. Afterward, expressed juices obtained from the PEF-treated cossettes at 20 °C and from the untreated ones at 80 °C were purified by dead-end ultrafiltration with stirring (500 rpm) at the temperature of 20 °C by using polyethersulfone membrane with MWCO of 30 kDa. Application of PEF (E?=?600 V cm^?1, t _PEF?=?10 ms, T?=?20 °C) with following pressing of cossettes at 5 bars during 15 min permits to obtain the juice yield Y?=?66,5 %, which is equivalent to that obtained from cossettes preheated to 80 °C and untreated electrically (Y?=?64 %). The energy consumption of cold PEF treatment (≈2–3 Wh/kg) is very attractive as compared to preheating at high temperatures (≈138–194 Wh/kg). Combination of thermal and electrical pretreatments leads to additional softening of sugar beet tissue and to a slightly higher (on 5–10 %) juice yield, but the electroporation of preheated cossettes is more energetically costly. The raw juice expressed from PEF-treated cossettes at 20 °C has higher purity (93.5 %) than juices expressed at 50 °C (92.9 %) and at 80 °C (92.3 %). The temperature increasing from 20 to 80 °C results in a higher juice coloration (5680 IU at 20 °C and 7820 IU at 80 °C) and leads to a higher (on about 35 %) colloids concentration in the expressed juice. The filtrate obtained from the juice expressed at 20 °C with PEF treatment has a higher purity (96 %) than the filtrate obtained from the juice expressed at 80 °C (95.3 %) and its coloration is considerably lower (330 IU versus 1930 IU). In addition, the quantity of proteins and colloids in the filtrate of juice expressed at 20 °C is lower than that in the filtrate of juice expressed at 80 °C