PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.     

Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell‐bound). Both enzymes were tested for their hydrolytic ability to p‐nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium‐ (C12) and long‐ (C18) chain fatty acids were used as substrates. Cell‐bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p‐nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p‐nitrophenyl decanoate > p‐nitrophenyl caprylate > p‐nitrophenyl laurate > p‐nitrophenyl palmitate > p‐nitrophenyl stearate. The cell‐bound lipase indicates preference for p‐nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell‐bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p‐nitrophenyl palmitate hydrolysis of cell‐bound lipase were significantly higher (K_m 2.462 mM and V_max 0.210 U/g/min) than those of extracellular lipase (K_m 0.406 mM and V_max 0.006 U/g/min). Copyright © 2010 John Wiley & Sons, Ltd.     

CHARACTERIZATION OF TRYPSIN PURIFIED FROM THE PYLORIC CAECA OF THE SOUTHWEST ATLANTIC WHITE CROAKER Micropogonias furnieri (Sciaenidae)

A southwest Atlantic croaker protease was purified from the pyloric caeca by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The enzyme was classified as a trypsin on the basis of molecular weight, its ability to hydrolyze synthetic substrates N-α-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and its inhibition by known trypsin inhibitors. The isolated enzyme has a single band on SDS-PAGE with an estimated molecular mass of 24 kDa. Croaker trypsin activity was stable between pH 5 and pH 11 for 30 min at 0C, 10C and 25C and its maximal activity against BAPA was at pH 9.5. Thermostability was observed to about 55C for 30 min at pH 7.8 and its temperature optimum was 60C. Substrate turnover number was 850 BAPA units per μmol trypsin, and the K_mwas 0.081 mM at 25C. For the hydrolysis of TAME, V_maxwas 9273 units per μmol trypsin and the K_m was 0.155 mM at 25C. The catalytic efficiency V_max/K_mwas higher than that of trypsin from fish living in colder waters.     

Comparison of the Proteolytic Activities of New Commercially Available Bacterial and Fungal Proteases toward Meat Proteins

The hydrolytic activity of 3 commercially available protease preparations (bacterial protease G, fungal 31000, and fungal 60000) were examined using fluorescent‐labeled casein, azo dye‐impregnated collagen, and meat protein extracts from bovine M. semimembranosus and Achilles tendon, and compared to that of papain. Assays showed that all proteases exhibited little activity at low temperature (5 °C), and maximal activity at 45 °C. The pH, at which optimal activity was observed for each of the protease preparations, differed and ranged from pH 5.0 to 8.0. Kinetic parameters (K_M and V_max) were also different between protease preparations, with the bacterial protease G and papain exhibiting significantly higher V_max values (P < 0.001) and lower K_M values (P < 0.01) for the casein substrate than the 2 fungal protease preparations. Meat protein hydrolysis was displayed on SDS‐PAGE and proteins analyzed with mass spectrometry. The protease preparations were shown to have varying affinity toward different meat proteins. The bacterial protease G preparation was efficient at hydrolyzing most myofibril and collagen proteins, and appeared to be more efficient than papain at hydrolyzing collagen proteins. On the other hand the 2 fungal protease preparations showed a selective specificity toward meat myofibrillar proteins, and the fungal 60000 protease preparation exhibited high affinity toward collagen γ and collagen type I chain B proteins. The results generated in this study demonstrated that these commercial proteases have good potential for use in meat tenderization applications due to their mild and complementary effects on different meat proteins. Practical Application : Bacterial and fungal protease preparations exhibited varying affinities for hydrolyzing meat proteins. This selective moderate capability of microbial proteases compared to papain is potentially an advantage in avoiding over‐tenderization in meat. On the other hand, the bacterial protease G preparation, which appeared to be more efficient at hydrolyzing connective tissue proteins than papain, could be beneficial in tenderizing meat with high connective tissue content. The synergistic effect of these protease preparations could be incorporated into a meat tenderizing formula to give the tenderizer a broad activity spectrum, thus able to target different cuts of meat.     

Properties of Trypsin from the Pyloric Ceca of Atlantic Cod (Gadus morhua)

Trypsin (EC 3.4.21.4) was isolated from the pyloric ceca of Atlantic cod and purified to homogeneity by affinity chromatography. The enzyme catalyzed the hydrolysis of benzoyl arginine p-nitroanilide (BAPA, pH 8.2 and 25°C) such that V_max was 250 BAPA units per micromole trypsin and K_m was 1.48 mM. For the hydrolysis of tosyl arginine methyl ester (TAME, pH 8.1 and 25°C), V_max was 18.2 × 10³ TAME units/micromole trypsin, and K_m 0.22 mM. The pH and temperature optima with BAPA substrate were 7.5 and 40°C, respectively. Atlantic cod trypsin was most active and stable at alkaline pH. The enzyme was heat labile, losing more than 50% of its activity after incubation at 50°C for 30 min. Amino acid analysis of Atlantic cod trypsin revealed that the enzyme was rich in residues such as serine, glycine, glutamate and aspartate, but poor in basic amino acid residues compared to trypsins from warm blooded animals.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

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.     

Controlled enzymatic hydrolysis of glycinin: Susceptibility of acidic and basic subunits to proteolytic enzymes

Glycinin, the major storage protein of soyabeans was enzymatically modified using papain, alcalase and fungal protease. The degree of hydrolysis (DH) was monitored by using trinitrobenzene sulphonic acid reaction with liberated α-amino groups. The DH could be varied by varying the ratio of enzyme to substrate, time and temperature of hydrolysis. The measured K_m and V_max values of glycinin with different proteases suggested that the susceptibility for hydrolytic cleavage of glycinin followed the order fungal protease>alcalase>papain. Electrophoretic analysis of cleaved glycinin suggested that acidic subunits of glycinin were cleaved preferentially over basic subunits. The measured K_m and V_max with acidic and basic subunits with fungal protease correlated with cleavage susceptibility. The functional properties of glycinin could be tailored by controlling the DH and using appropriate protease. Modified glycinin had better functional characteristics compared to glycinin.     

Kinetic Studies During Enzyme Hydrolysis of Potato and Cassava Starches

The hydrolysis of raw potato and cassava starches by bacterial α-amylase depends on the time of action, temperature and on the specific starch involved. The molecular weight of the trade α-amylase (Termamyl 60L), determined by SDS-PAGE, was found to be 55–65 kDa. The properties of α-amylase such as kinetic parameters, inhibition, stability, and thermostability were studied. The constants K_m and maximum reaction rate V_max for α-amylase were fitted to Michaelis-Menten models with these two starches. Differences in response of potato and cassava starches to hydrolysis by Termamyl 60L can explain differences found in K_m and V_max values and inhibition properties.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Aminopeptidase from jumbo squid (Dosidicus gigas) hepatopancreas: purification,characterisation, and casein hydrolysis

An aminopeptidase (AP) was partially purified from jumbo squid (Dosidicus gigas) hepatopancreas with 154.24‐fold and yield of 6.15%. The purification procedure consisted of ammonium sulphate fractionation and DEAE‐Sephacel chromatography. The enzyme was approximately 48–53 kDa as estimated by SDS‐PAGE. With l ‐leu‐p‐NA, it had optimum activity at pH 8.0 and 30 °C. The K_m and V_max/K_m values of the enzymes for l ‐leu‐p‐NA were 0.326 mm and 2787 at 37 °C, respectively. Activation energy (E_a) of the enzyme was 53.50 kJ M^?1.The AP showed activity against seven synthetic substrates: l ‐proline>l ‐methionine>Ac. l ‐γ‐glutamic>l ‐glycine>l ‐leucine>l ‐alanine>l ‐lysine‐p‐NA. The enzyme was strongly inhibited by Bestatin, partially inhibited by a metal‐chelating agent and by PCMB, a cystein protease inhibitor. Zn²⁺ and (or) Ca²⁺ seemed to be its metal cofactor(s). Incubation of casein with the partially purified AP resulted in a degree of hydrolysis of 6%.     

Pineapple fruit bromelain affinity to different protein substrates

Optimal pH and temperature conditions for proteolytic activity of pineapple fruit bromelain were determined using five different substrates: azocasein and azoalbumin (pH 3–10 at 20–70 °C), casein and sodium caseinate (pH 2–10 at 20–70 °C), and haemoglobin (pH 2–6.5 at 30–60 °C). Fruit bromelain has shown optimum activity at pH 7.5 for azoalbumin and at 6.5 for azocasein, all at 55 °C. Fruit bromelain activity determined with casein and sodium caseinate has shown optimum activity at 59 °C, while the optimum pH was 7.7 for casein and 6.5 for sodium caseinate. Optimum hydrolysis conditions of fruit bromelain towards haemoglobin showed a sharp peak at an acidic pH 2.9 at 37 °C. The lowest results of K_m and the highest results of V_max/K_m were found for azocasein and azoalbumin. These substrates are highly recommended for fruit bromelain activity determination.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

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.     

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose and d‐xylose

Genes encoding l ‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l ‐arabinose was dependent on a functioning l ‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d ‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l ‐arabinose showed that KmAxt1p has K_m 263 mm and V_max 57 nm /mg/min, and PgAxt1p has K_m 0.13 mm and V_max 18 nm /mg/min. Glucose, galactose and xylose significantly inhibit l ‐arabinose transport by both transporters. Transport assays using d ‐xylose showed that KmAxt1p has K_m 27 mm and V_max 3.8 nm /mg/min, and PgAxt1p has K_m 65 mm and V_max 8.7 nm /mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K_m 371 mm and V_max 341 nm /mg/min for l ‐arabinose, and K_m 25 mm and V_max 76 nm /mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l ‐arabinose and d ‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d ‐xylose or l ‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright © 2015 John Wiley & Sons, Ltd.     

PURIFICATION AND CHARACTERIZATION OF HOMOGENEOUS ACID PHOSPHATASE FROM NONGERMINATED BUCKWHEAT (FAGOPYRUM ESCULENTUM) SEEDS

A buckwheat acid phosphatase (orthophosphoric‐monoester phosphohydrolase, EC 3.1.3.2) was purified about 250‐fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p‐nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p‐nitrophenyl phosphate were determined to be K_M= 76 μmol L^?1 and k_cat= 924 s^?1 at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.     

EFFECT OF TWO POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION RELATIONSHIPS OF PROTEASES II. CHYMOSIN AND MUCOR MIEHEI PROTEINASE

The kinetics of chymosin and Mucor miehei proteinase (Mmp) catalyzed proteolysis of sodium (Na) caseinate, as well as their intrinsic structural properties, were examined in two polar organic-aqueous solvent systems. Chymosin and Mmp in standard buffer (10 mM phosphate, pH 6.0), 5% (v/v) ethanol (EtOH) in standard buffer and 5% (v/v) acetonitrile (ACN) in standard buffer were used in the investigation. Kinetic parameters for the hydrolysis of Na-caseinate by chymosin showed that, relative to standard buffer, the K_m in both 5% EtOH in standard buffer and 5% ACN in standard buffer increased significantly (p ≤ 0.05) to nearly the same level from 4.8 mg/ml to 7.5 mg/ml and 7.7 mg/ml, respectively. No significant changes (p > 0.05) were observed in V_max in 5% EtOH (12.8 μmoles/min.mg) or 5% ACN (11.8 μmoles/min.mg) compared with standard buffer (12.8 μmoles/min.mg) resulting in V_max/K_m values reduced to a similar extent in both organic solvents. For Mmp, a glycoprotein, K_m increased (p ≤ 0.05) in 5% ACN in standard buffer (5.3 mg/ml, yet it decreased (p ≤ 0.05) in 5% EtOH in standard buffer (3.1 mg/ml) compared with standard buffer (4.2 mg/ml). The solvent-induced decrease in V_max for Mmp was somewhat larger in 5% EtOH in standard buffer than 5% ACN in standard buffer, from 17.3 μmoles/min.mg (standard buffer) to 14.0 μmoles/min.mg (p ≤ 0.05) in 5% EtOH in standard buffer but was not significantly different (p > 0.05) in 5% ACN in standard buffer (15.7 μmoles/min.mg). For both chymosin and Mmp, changes in kinetic parameters appeared to correspond with solvent-induced structural changes as evidenced by near-UV circular dichroism (CD) spectroscopy; higher K_m corresponded to a higher ellipticity in the near-UV CD spectra (240–320 nm), which may indicate a decreased protein flexibility. The fact that chymosin and Mmp behaved differently in these organic solvents indicates that factors other than polarity of the media may also have been involved in this phenomenon since both 5% EtOH in standard buffer and 5% ACN in standard buffer solutions were of the same polarity based on E_T(30) scale. Different specificities for Na-caseinate hydrolysis between chymosin and Mmp, as well as altered specificities of both enzymes for the substrate caused by organic solvents, were demonstrated in SDS-PAGE peptide maps as differences in banding patterns. Differential scanning calorimetric studies on chymosin and Mmp in the two solvent systems showed destabilization (lowered temperature of denaturation) of the enzymes in both 5% EtOH in standard buffer and 5% ACN in standard buffer relative to standard buffer.     

EFFECT OF TWO POLAR ORGANIC-AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE-FUNCTION RELATIONSHIP OF PROTEASES I. PEPSIN

The effect of various organic-aqueous solvent systems on the kinetic parameters, intrinsic spectral properties, thermal stability, and proteolytic patterns of porcine pepsin were studied. Two substrates, Z-His-Phe(-NO₂)-Phe-OMe and sodium (Na-) caseinate, were chosen. Three different buffer compositions were used in the investigation: (1) the standard buffers (20 mM formate buffers, pH 2.1 and 10 mM phosphate buffer, pH 5.7); (2) 5% (v/v) ethanol (EtOH) in the standard buffers; (3) 5% (v/v) acetonitrile (ACN) in the standard buffer. Relative to pepsin in formate buffer (pH 2.1), the K_m for Z-His-Phe(-NO₂)-Phe-OMe in 5% EtOH increased from 0.57 mM to 1.03 mM (p ≤ 0.05), while no significant difference was observed in 5% ACN (p > 0.05). The solvent-induced decrease in V_max was much larger in 5% ACN than in 5% EtOH, from 48.0 nmoles/min.mg to 12.3 nmoles/min.mg (p > 0.05) and 35.0 nmoles/min.mg (p > 0.05), respectively, as compared to standard buffer. Relative to pepsin in 10 mM phosphate buffer (pH 5.7), the K_m for Na-caseinate in both 5% EtOH and 5% ACN increased from 4.1 mg/ml to 7.8 mg/ml and 6.2 mg/ml (p ≤ 0.05), respectively while only 5% ACN caused a significant decrease in V_max compared with standard buffer, from 11.8μmoles/min.mgto 7.6 μmoles/min.mg (p ≤ 0.05). Changes in kinetic parameters generally corresponded with solvent-induced structural changes as evidenced by circular dichroism (CD) spectroscopy, a low K_m corresponding to low ellipticity in the near-UV CD spectra (240–320 nm) possibly indicative of a greater protein flexibility. The above results were attributed to differences in properties other than polarity of the solvent systems since the polarity of both 5% EtOH and 5% ACN solutions, as measured by E_T(30) scale, was the same. Differential scanning calorimetric studies of pepsin in the different solvent systems showed destabilization of the enzyme in the organic solutions relative to standard buffer, i.e., lowered temperature of denaturation. Altered specificity of pepsin for Na-caseinate hydrolysis in the presence of the various organic solvents was demonstrated in SDS-PAGE peptide maps as differences in the banding patterns.     

STUDY OF THE OXIDATION OF RESVERATROL CATALYZED BY POLYPHENOL OXTOASE. EFFECT OF POLYPHENOL OXIDASE, LACCASE AND PEROXIDASE ON THE ANTIRADICAL CAPACITY OF RESVERATROL

This paper reports the study of the oxidation of resveratrol catalyzed by polyphenol oxidase (PPO) as ‐well as the effect of PPO, laccase and peroxidase on the antiradical capacity of resveratrol (measured as capacity to scavenge 2,2′‐azino‐bis (3‐ethylbenzthiazoline ‐ 6 ‐ sulfonic acid) radicals (ARTS')). Monophenolase activity of mushroom PPO in the presence of resveratrol showed the characteristic lag period (τ) prior the attainment of the steady state rate (V_ss). The Michaelis constant (K_m) for the oxidation of resveratrol catalyzed by PPO was 45 ± 2 μM at pH 6.8 and it decreased with pH. However, the maximum steady state rate (V_max) remained constant with pH. Both τ and V_ss depended on pH in a sigmoid way. Kinetic and NMR assays confirmed that this compound is a substrate which fulfilled both kinetic and structural reaction mechanisms of PPO. Neither laccase, nor PPO modified the antiradical capacity of resveratrol. However, resveratrol, in the presence of peroxidase, lost its antiradical capacity. A possible correlation between antiradical capacity of resveratrol and its oxidation status is proposed. This paper tries to increase the knowledge about this promising health‐beneficial molecule and to demonstrate that PPO could be involved in the oxidation pathway of resveratrol.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.