Rennin-like milk coagulant enzyme produced by a local isolate of Mucor

Among 20 isolates of Mucor isolated from various environments in Jordan and found to produce a rennin-like acid protease, known as Mucor rennin-like enzyme (MRE), Mucor J20 was found to produce the highest level of MRE. The optimum incubation conditions for enzyme production in a fortified wheat bran mixture using solid-state fermentation were 3–4 days at 30°C. The highest MRE activity (185–200 rennin units or RU) was produced in a medium containing wheat bran and lentil straw (1 : 1 w/w) moistened with whey, and incubated in clay pots at 30°C for 4 days. A slightly lower activity value (178 RU) was found when using a mineral salt solution or distilled water instead of whey, or when using wheat bran alone with whey. At pH 4, the MRE retained its complete activity (100%) for 6 weeks at 5°C and 10°C, and for 3 and 2 weeks at 20°C and 30°C, respectively. After heating at 60°C for 10 min, the enzyme lost its activity at all pH levels used (pH 2–8). The crude extract of MRE was successfully applied in the manufacture of a cheese curd.     

Storage Effects on Lipase Activity in Fresh-cut Cantaloupe Melon

ABSTRACT: Lipase enzyme in fresh-cut cantaloupe melon ( Cucumis melo L. var. reticulatus Naud) was assayed, and the effects of storage at 4 and 15 °C on enzymatic activity were determined. The enzyme was stable for 24 h at both temperatures but decreased by 45% after 120 h of storage time at 15 °C. Isoelectric studies suggest a possible dual lipase and esterase action of the enzyme. Enzymatic activity increased by about 60% when the reaction temperature was increased from 30 °C to 40 °C and remained constant until 70 °C, but the enzyme was unstable when stored at the high temperature. Calcium treatment reduced lipase activity in the fresh-cut fruit.     

Encapsulation and stimulated release of enzymes using lecithin vesicles

Lecithin vesicles prepared by dehydration-rehydration (DR) were used to encapsulate enzymes (lysozyme and pepsin). The encapsulating efficiency was highest when the pH was close to the isoelectric point of each enzyme. Vesicles stored in suspension at 10°C for up to 21 days showed no release of enzymes. Acidic pH at 10°C and 25 mM Ca²⁺ at 10°C or 37°C produced pulse-like release of 17–35%, while acidic pH at 37°C produced pulse-like release followed by slow release up to 100%, and Tween 80 induced steady release from the beginning. The hydrolysis pattern of a protein by pepsin released from DR vesicles for 142 hr was similar to that obtained by the same total amount of fresh pepsin solution added stepwise, in proportion, indicating that the pepsin retains its activity throughout the period of encapsulation.
Vesicles prepared by processing of lecithin-enzyme solution by a homogenizer (Microfluidizer^TM) were also characterized and found effective.     

Partial characterization of the in situ activity of pectinesterase in Bramley apple

The characteristics of pectinesterase (PE) have been examined in waste material (peel, cores and offcuts) obtained from Bramley seedling apples barn stored for 2, 4 and 12 weeks. A crude enzyme extract was prepared by suspending the dried and milled apple waste in 0.1 m NaCl at pH 8.5. The activity of PE under standard assay conditions of 0.5% apple pectin, 0.1 m NaCl, pH 8.5 and 30°C was low, between 4 and 8 units g⁻¹ dry matter, but activities up to 60 units g⁻¹ dry matter were obtained at higher temperatures. The optimum temperature was 60°C with the enzyme stable up to 40°C with 5 min heating. The mean activation energy for PE in the three samples was calculated at 39.2 kJ mol⁻¹ K⁻¹. The optimum pH was high at 10.0 probably due to the PE assay measuring the extraction/solubilization and stability of the enzyme in addition to its activity. Optimum activity was obtained in 0.15 m NaCl with optimum stability at 0.5 m.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

The influence of water activity on thermal stability of horseradish peroxidase

The thermal stability of horseradish peroxidase in the solid state was studied as a function of water activity, from 0.11 to 0.88. At all activities the enzyme was found to be much more stable in the solid state than in solution. Inactivation temperatures were in the range of 140–160°C. Inactivation curves show a biphasic behaviour which can be described by a model assuming two fractions (heat labile and heat stable) with independent first order inactivation kinetics. The labile fraction represents approximately 30% of the total activity. The z-value for both stable and labile fractions depends on water activity (moisture content) and has a maximum at a_w= 0.76 (44.4°C and 43.8°C, respectively).     

Extracellular protease from Mucor pusillus: purification and characterization

Extracellular protease from Mucor pusillus was purified 18-fold with 7.56% recovery by ion-exchange chromatography and gel filtration. The enzyme was found to be monomeric in nature, having a molecular mass of 49 kDa. The enzyme acted optimally at 50°C and was stable in the temperature range 30–50°C. It was completely inactivated by heating for 30 min at 65°C. The optimum of activity for the purified extract was observed at milk CaCl₂ concentration of 0.02  m and at milk pH of 5. These properties, except for temperature, were similar to those of rennet.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

Purification and Characteristics of Feruloyl Esterase from Aspergillus awamori G-2 Strain

ABSTRACT:  For food industry production processes and other uses, a mold that produces high levels of feruloyl esterase was obtained from laboratory mold collections and other sources. It was Aspergillus awamori G-2 that produces high levels of feruloyl esterase. The feruloyl esterase was purified using ion-exchange chromatography, size-exclusion chromatography, and HPLC chromatography. The enzyme was identified as a monomer protein using size-exclusion chromatography. Its optimum temperature and pH were, respectively, 40 °C and pH 5. Its activity was stable at pH 3 to 5. The enzyme was combined with xylan and starch, but it was absorbed by cellulose. The km of the feruloyl esterase was 0.0019% (0.01 mM). The enzyme showed stable activity at pH 3 and 50 °C, making this enzyme useful for food production.     

Stability of β-glucosidase Activity Produced by Bifidobacterium and Lactobacillus spp. in Fermented Soymilk During Processing and Storage

ABSTRACT: Microorganisms possess endogenous enzymes, however the stability of these enzymes during storage in soymilk has not been studied. β-glucosidase is an important enzyme that could be used in the bioconversion of the predominant soy isoflavone glucosides to their bioactive aglycone forms. Fifteen probiotic microorganisms including bifidobacterium, Lactobacillus acidophilus , and Lactobacillus casei were screened for β-glucosidase activity using p-nitrophenyl-β-d-glucopyranoside as a substrate. Six strains were selected on the basis of β-glucosidase activity produced during fermentation of soymilk. The stability of the enzyme activity was assessed during incubation for up to 48 h and storage for 8 wk at frozen (-80°C), refrigerated (4°C), room (24.8°C), and incubation (37°C) temperatures. L. casei strains showed the highest β-glucosidase activity after 24 h of incubation followed by L. acidophilus strains, whereas bifidobacterium strains showedleast activity. However, p-glucosidase from Bifidobacterium animalis BB12 showed the best stability during the 48 h fermentation. Lower storage temperatures (-80°C and 4°C) showed significantly higher ( P < 0.05) β-glucosidase activity and better stability than that at higher temperatures (24.8°C and 37°C). The stability of β-glucosidase from these microorganisms should be considered for enzymic biotransformation during storage of isoflavone β-glucosides to bioactive isoflavone aglycone forms with potential health benefits.     

Characterization of Low Saturation Palm Oil Products after Continuous Enzymatic Interesterification and Dry Fractionation

ABSTRACT:  The lipase-catalyzed interesterification of refined, bleached, deodorized palm olein with iodine value (IV) of 62 was studied in a pilot continuous packed-bed reactor operating at 65 °C. Sn- 1,3 specific immobilized enzyme; Lipozyme® TL IM ( Thermomyces Lanuginosa ) from Novozyme A/S was used in this study. The interesterification reaction produced fully solidified fats at ambient temperature due to the production of trisaturated triacylglycerols (TAG) (PPP and PPS, where P = palmitic acid, S = stearic acid). The reaction also increased the percentage of triunsaturated TAG (OLL, OLO, and OOO, where O = oleic acid, L = linoleic acid). The interesterified product was then dry fractionated at temperatures of 9, 12, 15, 18, and 21 °C to separate the saturated fats from the unsaturated. The results show that IV of olein increased when the fractionation temperature (T_FN) decreased. The highest IV of olein was 72, obtained from T_FN at 9 °C. After interesterification and laboratory-scale fractionation, the olein fractions contained higher unsaturation content ranging from 64.7% to 67.7% compared to the starting material (58.3%), while the saturation content was reduced from 41.7% to the range of 32.3% to 35.3%. The yields of these oleins were low with the range of 24.8% to 51.8% due to the limitation of the vacuum filtration. Ten kilograms of pilot-scale fractionation with membrane press filter was used to determine the exact olein yield. At T_FN of 12 °C, 67.1% of olein with saturation content of 33.9% was obtained.     

Enhanced deactivation of bacterial lipases by a modified UHT treatment

Lipase was produced by three non-fluorescent pseudomonads during culturing in resterilized UHT whole milk at 10°C. The enzymes exhibited pronounced thermostability in milk with 85–88 and 82–89% of the original activities retained after heat treatments at 140°C for 5 sec and 80°C for 10 min, respectively. Also, after a single treatment at 60°C for 10 min approximately 95% of the untreated activities remained.
Double heat treatments consisting of either 130 or 140°C for 5 sec followed by 60–80°C for 3 min enhanced lipase deactivation by as much as 40% compared with the combined effect of both higher and lower temperature treatments performed on separate enzyme samples. No significant enhancement of deactivation was noted when samples were heated at 60°C for 3 min followed by 140°C for 5 sec compared with the latter treatment alone.
Lipase deactivation was non-linear at 60°C following treatment at 140°C for 5 sec; no substantial additional loss of activity occurred at the lower temperature between 5 and 10 min.     

NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation

ABSTRACT:  Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 °C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca²⁺ activated the enzyme activity but reduced enzyme stability at 65 °C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.     

KINETIC PARAMETERS FOR THERMAL INACTIVATION OF PLASMIN

Plasmin (EC 3.4.21.7) is the principal indigenous protease in bovine milk. Kinetic parameters for thermal degradation of plasmin were determined using a miniature continuous flow pasteurizer designed for heat treatment of small quantities of liquid. Plasmin activity was measured using a pH-stat titration, which measures the release of H ⁺ from a caseinate substrate.
Heating milk at various temperatures resulted in significant difference (P > 0.01) in residual plasmin activity and linear decrease in activity at each temperature. The calculated D-values were: 105, 90, 76.5, 62, and 47 s at 72, 78, 85, 92, and 100°C, respectively. The Z-value for plasmin inactivation was estimated to be 77.5°C, and the Arrhenius activation energy was 7.04 kcal/mole.
UHT milk containing plasmin was heated for an equivalent of 5 D inactivation of the enzyme at 100°C. After storage for 30 days at 4°C, no enzyme regeneration was observed.     

PREPARATION AND CHARACTERIZATION OF α-AMYLASE IMMOBILIZED ON COLLAGEN MEMBRANES

Crystalline pancreatic α-amylase was codispersed with hide collagen at pH 4.0 and tanned to form a membrane which degraded starch. The optimum pH for the codispersed membrane preparation was at pH 7.0 in contrast to the soluble enzyme which was as active at pH 8.0 as at pH 7.0. The immobilized enzyme responded maximally to 0.22M chloride whereas 0.02M chloride gave optimum rates for the soluble enzyme. The immobilized enzyme resisted thermal inactivation better than the soluble α-amylase. Raising the temperatures from 30 ° to 50 °C produced a 500% increase in rate for the bound enzyme. It was also demonstrated that membranes retained greater activity when stored in starch solution than in water. The effect of glutaraldehyde concentration on membrane activity was also studied.     

Seasonal changes and preliminary characterization of cathepsin D-like activity in sardine (Sardina pilchardus) muscle

The seasonal changes of cathepsin D-like activity in white and dark muscle of sardine were followed and maximum activity was found in April and October, which could be related to the spawning period of this species. Higher catheptic activity was measured in dark muscle and females. The crude extract of this muscle enzyme had an optimum pH and temperature of 3.2 and 55 °C, respectively, and the activity was strongly inhibited by low levels of NaCl (6%). The thermal stability was lower at pH 3.2 than at pH 6.5 and the enzyme was stable in the pH range 3–4.5. The activity of this protease was detected during the ripening process of salted sardine which suggests its participation in this process.     

Heat-induced Activation of Polyphenoloxidase in Western Rock Lobster (Panulirus cygnus) Hemolymph: Implications for Heat Processing

ABSTRACT: Polyphenoloxidase (PPO) activity in western rock lobster (WRL) hemolymph results in blackening, or melanosis , of the tissues after processing. The impact of processing temperatures on WRL PPO activity was evaluated under steady state and non-steady state temperature conditions. Baseline PPO activity and total PPO activity were determined spectrophotometrically. PPO activity showed a heat-induced net activation effect between 60 and 80 °C. Deactivation of the enzyme was not significant until temperatures reached 90 °C. During heating, a balance existed between heat-induced activation and deactivation of PPO. Therefore, maximization of PPO deactivation and minimization of melanosis formation in processed WRL would require the internal temperature of processed lobsters to exceed 90 °C.     

Influence of Extrusion Processing on Procyanidin Composition and Total Anthocyanin Contents of Blueberry Pomace

ABSTRACT:  Blueberry juice processing by-products are a rich source of procyanidins, which comprise a group of compounds shown to possess numerous health benefits, including protection against coronary heart disease, type II diabetes, and obesity. Most of the procyanidins present in blueberry pomace, however, are large molecular weight compounds that are poorly absorbed and show weak bioactivity compared to the smaller molecular weight monomers and dimers. The objective of our study was to identify optimal extrusion variables to enhance the contents of monomers and dimers at the expense of large molecular weight procyanidin oligomers and polymers. Extrusion variables temperature (160 and 180 °C) and screw speed (150 and 200 rpm) were tested using mixtures of blueberry pomace with decorticated white sorghum flour at a ratio of 30 : 70 and 45% moisture content. Extrudates were analyzed for procyanidin composition and total anthocyanin content. Extrusion of blueberry pomace increased the monomer, dimer, and trimer contents considerably at both temperature and screw speeds. The highest monomer content, obtained at 180 °C and 150 rpm screw speed, was 84% higher than the nonextruded control. Significantly higher levels of dimer and trimer contents were also obtained under these conditions. Increases in monomer, dimer, and trimer contents apparently were the result of reduced polymer contents, which was approximately 40% lower for samples extruded at 180 °C temperature and 150 rpm screw speed. Extrusion processing reduced total anthocyanin contents by 33% to 42% indicating that additional treatments are needed to retain the pigments. These results demonstrate that extrusion processing can be used to increase procyanidin monomer and dimers in blueberry pomace.     

Pectin Methyl Esterase Activity in Southern Peas (Vigna sinensis)

SUMMARY: Pectin methyl esterase (PME) activity was investigated in vitro in Southern peas (Vigna sinensis). Experiments were conducted to determine the effect of NaCl concentration, pH, temperature, maturity, frozen storage and rinsing the peas on PME activity.
The optimum salt level for maximum PME activity in the Purple Hull Pink Eye (PHPE) and Princess Ann (PA) varieties was ascertained to be 0.25 M. The pH optima were: PHPE, 8.5; and PA, 7.5 to 8. The remaining studies were made using peas of the PHPE variety which were harvested at 3 stages of maturity. Maximum enzymatic activity occurred at 50°–60°C. Partial inactivation occurred at 65°C. The mean Q₁₀ was 1.35 over the range of 30°–50°C.
PME activity was dependent upon the maturity of the peas. The most immature peas had an activity level about 2.5 times that found in the most mature peas. Peas subjected to frozen storage had a higher activity than the fresh peas; the increased activity was more pronounced in the more mature peas.
Rinsing the peas removed significant amounts of the enzyme. A greater proportion of PME was removed from the more immature peas than from the more mature ones. With the immature, frozen peas PME activity was reduced 22.7% by rinsing; whereas, in the fresh counterpart the reduction was 11.1%. The rinse water from frozen peas contained more PME activity than did rinse water from fresh peas. Also, the activity in rinse water from the most immature peas was about 2.7 times that from the most mature ones. Upon standing, PME activity in the slurries prepared from frozen peas continued to increase up to 3–4 hr.     

Potential Application of Hot Rehydration Alone or in Combination with Hydrogen Peroxide to Control Pectin Methylesterase Activity and Microbial Load in Cold-stored Intermediate-moisture Sun-dried Figs

ABSTRACT: Sun-dried figs contain a considerable amount of pectin methylesterase (PME) activity (22 JAM COOH/ min/g). The enzyme causes softening and loss of desired gummy texture in cold-stored intermediate-moisture (IM) sun-dried figs brought to a 28% to 29% moisture range. Partial reduction of PME activity (28%) delayed undesirable textural changes in IM figs rehydrated at 80 °C for 16 min. The heat treatment did not cause a considerable reduction in microbial load. However, the addition of 2.5% H2O2 to the rehydration medium at 80 °C reduced the initial total mesophilic aerobic count of figs by at least 90% and turned the figs from a brown color to a desirable and stable yellow-light brown. The in situ fig catalase remains after rehydration at 80 °C. Thus, by reducing the contact period of figs with H2O2 or by pureeing figs, it is possible to eliminate residual H2O2 and to obtain safe and SO2-free light-colored fig products.