SCREENING FOR PECTINOLYTIC CANDIDA YEASTS: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYMES

In screening 72 Candida strains for extracellular enzymes, one acidic and seven alkaline proteases, one amylase and six pectinases were found. Candida kefyr, Candida macedoniensis and four strains from Candida pseudotropicalis were pectinolytically active; Candida kefyr showed considerable activity also under aerobic conditions but the highest activities were attained under strictly anaerobic conditions with N₂ gassing. YNB and peptone were the best nitrogen sources as regards enzyme production. The highest enzyme activities were achieved with succinic acid (aerobic) and inulin (anaerobic) as carbon sources. Enzyme production under aerobic conditions was considerably increased at pH 3.0. The addition of inducers (pectins or pectin ballast substances) led only to very slightly increased enzyme production. From kinetic studies of the enzyme, optimum activity was found to lie at pH 5.0 and 50°C. Subsequent characterization of the enzyme showed it to be an endo-polygalacturonase. Eight to ten bands could be resolved by analytical isoelectric focussing. The K_m value was found to be 1.14 × 10^?5 mol. The maceration test, using potato, carrot and apple tissue as substrate, showed considerable maceration activity, especially in the case of apple.     

Partial purification and characterization of peroxidase from olives (<Emphasis Type="Italic">Olea europaea</Emphasis> cv. Koroneiki)

The enzyme peroxidase (POD) activity was extracted from olives (Olea europaea cv. Koroneiki) and was partially purified by ammonium sulfate fractionation and gel permeation chromatography (Sephacryl S 300). Further characterization of the POD was performed using the ammonium sulfate purified fraction. POD showed a molecular mass of 44 ± 2 kDa and it expressed catalytic activity with 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylenediamine (DMPD) and some olive fruit phenols. However, the enzyme was found ineffective as regards the oxidation of oleuropein, the major polyphenol of olives, as well as with coumaric, ferulic, ascorbic and p-hydroxy benzoic acids. pH optimum of the peroxidase-catalyzed oxidation depended on the substrate used and it varied from 4.0 to 6.0. Olive peroxidase shows high thermal stability. Oleuropein, the major polyphenol of olives, drastically inhibited ABTS peroxidation by the POD preparation with an IC₅₀ value of 47 μM. The presence of POD enzyme activity in virgin olive oil samples was also confirmed.     

THE CHARACTERISTICS OF SOYBEAN PHYTASE

Soybean phytase was extracted with 2% CaCl₂ and partially purified by ammonium sulphate fractionation followed by dialysis in 0.01 M tris-maleate buffer, pH 6.5. The enzyme showed an optimum pH of 4.8 and optimum temperature of 60°C. The phytase was partially inhibited at high substrate concentration, with an optimum substrate concentration at 20 mM and a K_m value of 2.4 × 10^-3 M. V_max was 0.22 μmole P_i liberated/min/mL enzyme. The inactivation and activation energies for the hydrolysis of phytic acid were approximately 47,000 cal/mole and 11,100 cal/mole, respectively. Enzyme activity was inhibited by about 25%, 23% and 22% in the presence of 10^-3 M Zn⁺⁺, Cu⁺⁺ and Hg⁺⁺, respectively, and was also decreased by about 85% in the presence of 10^-1 M N-ethylmaleimide and sodium fluoride. Reducing and chelating agents at concentrations up to 10^-1 M inhibited activity by about 50% and by more than 90%, respectively.     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Purification of New β-Galactosidase from Enterococcus faecium MTCC 5153 with Transgalactosylation Activity

A new β-galactosidase (β-gal) was purified from a lactic acid bacterial strain of Enterococcus faecium MTCC5153 by chromatographic techniques. The purified enzyme had a specific activity of 24.06 U/mg of protein with k _m and V_max values of 2 mM and 18.2 mM/min/mg of protein, respectively. The yield of purified β-gal was 10.65% and estimated molecular weight found to be ～90 kDa, consisting of two homodimeric subunits of 43kDa. The enzyme was stable in pH range of 8.0–9.0 with an optimum pH of 8 and the optimum temperature of 40°C. The enzyme was activated in the presence of metal ions such as Mg⁺², Mn⁺², Ca⁺², K⁺ and Na⁺ and was inhibited by Zn⁺², Co⁺² and Cu⁺². Chemical modifiers (N-bromosuccinamide and Diethylpyro carbonate) inactivated the enzyme indicating the role of tryptophan and histidine moieties for activity. The purified β-gal was able to synthesize oligosaccharides from lactose. This study suggests that the β-gal of Enterococcus faecium MTCC5153 could be applied in dairy industry for hydrolysis of lactose and to improve its digestibility. β-gal of probiotic cultures are of particular interest due to their transgalactosylation properties.     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry     

Characterization of purified xylanase from finger millet (<Emphasis Type="Italic">Eleusine coracana-</Emphasis>Indaf 15) malt

Xylanase (E.C. 3.2.1.8) was purified to apparent homogeneity from 96 h finger millet (Eleusine coracana, Indaf-15) malt by a three step purification procedure via ammonium sulphate fractionation, DEAE-cellulose ion exchange and Sephadex G-75 gel permeation chromatographies with a recovery of 4.0% and fold purification of 60. Xylanase, having a molecular weight of 29 ± 2 kDa was found to be monomeric on SDS-PAGE. pH optimum of the enzyme was found to be in the range of 5.0–5.5. The activation energy was 25 kJmol⁻¹. Xylanase showed maximum stability at 35 °C in a pH range of 5.0–6.0. K _m and V _max of purified xylanase were found to be 0.2% and 4.5 μmol min⁻¹, respectively. Metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ag²⁺ and Ni²⁺ enhanced xylanase activity at 5 mM concentration. p-chloromercuribenzoate, citric, oxalic and boric acids inhibited the enzyme in concentration dependent manner. The mode of action of xylanase was found to be “endo” as determined by the analysis of products liberated from larchwood xylan by ESI-MS and H¹NMR. In vitro studies using Bifidobacterium and Lactobacillus sp. confirmed the prebiotic activity of the xylo-oligosaccharides.     

ALPHA-GALACTOSIDASE OF GERMINATING SEEDS OF CASSIA SERICEA SW.

Germinating seeds of Cassia sericea Sw. contain two molecular forms of α-galactosidase which were partially purified and characterized. Both enzyme forms had an optimum pH of 5.0 and an optimum temperature of 50 °C. K_m values for the substrate p-nitrophenyl-α-D-galactoside (PNPG) were 0.91 mM and 1.05 mM for the two forms, and substrate inhibition was observed at high concentrations of PNPG. The two forms of the enzyme had molecular weights of 46,000 and 33,000 by gel filtration. The enzyme forms were inhibited completely by Ag⁺, Cu²⁺ and Hg²⁺ ions and by p-chloromercuribenzoate showing that thiol groups are probably involved in catalysis. Both α-galactosidases hydrolyzed melibiose and raffinose, although hydrolysis of stachyose could not be detected. The enzyme may find potential use in the food industry to hydrolyze flatulence-causing sugars in processed foods and in production of sucrose from high-raffinose sugar beets. That the source (C. sericea seeds) of the enzyme is inexpensive provides an added advantage over use of cultivated legumes as a source of α-galactosidase.     

OPTIMIZATION OF THE LACTIC ACID FERMENTATION OF HYDROLYZED COD (GADUS MORHUA) GURRY WITH CORN SYRUP AS CARBOHYDRATE SOURCE

ABSTRACT The purpose of this study was to determine the optimum concentrations of corn syrup required with the use of several species of homofermentative lactic acid bacteria and a commercial mixed culture inoculum in the fermentation of hydrolyzed cod gurry. Among the three homofermentative bacteria studied, Lactobacillus plantarum, Streptococcus faecium, and Pediococcus acidilactici, consistent results regarding the most rapid rate of pH reduction and the lowest final pH were obtained with L. plantarum. S. faecium was the least desirable organism on the basis of fermentation rates and the extent of pH reduction after 72 h of incubation. Corn syrup at a concentration of 4.0% (w/w) was found to be optimum for achieving a rapid and successful lactic acid fermentation of hydrolyzed cod gurry with Lactobacillus plantarum. Corn syrup at a concentration of 4.5% was found to be optimum for rapidly achieving a final pH of 4.0 with the commercial mixed inoculum Stabisil. The maltose content of corn syrup was utilized at a notably slower rate than glucose by all three microorganisms during the first 18 h of incubation. Throughout the fermentations, maltotriose was utilized significantly by only L. plantarum.     

CHARACTERIZATION OF AN 'ENDOPOLYGALACTURONASE' OF THE YEAST KLUYVEROMYCES MARXIANUS

Analytic isoelectric focusing showed that the ‘endopolygalacturonase’ from Kluyveromyces marxianus consists of 21 multiple enzyme components. Neither changes in cultivation conditions, enzyme substrate or reaction conditions resulted in any quantitative or qualitative differences in the enzyme patterns. Two of these multiple enzyme forms, the main band, (IP = pH 5.8) which accounted for approximately 95% of the total activity, and an ‘acid-band’ (IP = pH 2.7), were purified by means of preparative isoelectric focusing and their molecular weights and amino acid compositions were determined. The molecular weight of the main band was established as 76,000 daltons (2 subunits of 47,900 and 28,100), The molecular weight of the ‘acid-band’ was 32,200. Both amino acid analyses and molecular weight determinations suggested that the proteins are chemically and physically different. The pH optimum was 4 for the pure enzyme on different pectin substrates, e.g., high molecular weight pectic acid, low molecular weight pectic acid and highly methylated pectins B and C. The temperature optimum obtained for pure enzyme with high or low molecular-weight pectic acid as substrate was 40° C. V_max and Km-value determination at different pH values with low molecular weight pectic acid as a substrate was used to identify the catalytically active groups at the active site. They were tentatively identified as an unprotonated α-carboxyl group and a protonated carboxyl group of aspartic acid.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

CHARACTERIZATION OF AFFINITY-PURIFIED TRYPSIN FROM HYBRID TILAPIA (TILAPIA NILOTICA/AUREA)

Trypsin was extracted from the intestines of tilapia and purified 136-fold on the basis of amidase activity by a sequence of ammonium sulfate fractionation, acetone precipitation and affinity chromatography on soybean trypsin inhibitor bound to 4.0% beaded agarose. The purified trypsin exhibited higher esterase than amidase activity at its optimum pH of 9.0. The enzyme was stable in the range of pH 7–9. The optimum temperature for enzyme activity was 40C and the Q₁₀ for enzyme activity was 1.7 from 20C–30C and 30C–40C. The Arrhenius energy of activation was 8.9 and 8.7 kcal./mol respectively, for the ranges of 20C–30C and 30C–40C.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

STABILITY AND PROPERTIES OF A THERMOSTABLE β-GALACTOSIDASE IMMOBILIZED ON CHTTIN

Thermostable β‐galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized β‐galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively.     

Selection of lactic acid bacteria for acidification of brown juice (grass juice), with the aim of making a durable substrate for L‐lysine fermentation

BACKGROUND: Brown juice is a waste product from the green crop‐drying industry. Heating and pressing of green crops prior to drying produces a juice rich in nutrients, which can be converted to a stable universal fermentation medium by lowering its pH to 4.0–4.5 via lactic acid fermentation. The aim of this study was to select a strain of lactic acid bacteria for industrial acidification of brown juice. RESULTS: Several strains were tested in fermentation experiments and Lactobacillus salivarius ssp. salivarius DSM 20555 was found to be the best choice. It showed high growth rates at temperatures ranging from 40 to 48 °C, with an optimum temperature of 46 °C (µ_max = 2.2 h^?1), and maintained a high productivity at pH 4.0 in continuous fermentation. The highest productivity of 7.23 g L^?1 h^?1 was found at a dilution rate of 1.0 h^?1. CONCLUSION: With today's increased focus on utilisation of residues from food and agro‐industry, coupled with restrictions on their use as fertiliser for farmlands, lactic acid fermentation could play a significant role in conservation of these nutrient‐rich liquids. This study shows that Lb. salivarius ssp. salivarius DSM 20555 is a very promising micro‐organism for use in such a process. Copyright © 2008 Society of Chemical Industry     

Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16

A novel phytase from thermophilic Geobacillus sp. TF16 was puri?ed approximately 5-fold using ammonium sulfate precipitation and ion exchange chromatography, and determined as a single band 106.04 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature and optimum pH were found to be 85°C and 4.0, respectively. The enzyme is highly thermostable and V_max and K_m values were calculated as 526.28 U/mg and 1.31 mM, respectively. It was also found that the enzyme exhibited a broad substrate selectivity and resistance toward proteases and effectively hydrolyzed soymilk phytate. These results suggest that this study provides an alternative phytase enzyme with enhanced properties.     

Characterisation of an anionic peroxidase from horseradish cv. Balady

An anionic peroxidase POIII, molecular weight 56 kDa, was purified from the roots of horseradish cv. Balady. The enzyme exhibited high activity towards o-phenylenediamine and guaiacol, while o-dianisidine had moderate peroxidase activity. Pyrogallol and p-aminoantipyrine had low affinity toward POIII. POIII was found to have a temperature optimum at 40 °C; the enzyme activity remained stable up to 40 °C and retained 87%, 51% and 29% of its activity at 50, 60 and 70 °C, respectively. The enzyme exhibited more than 50% of activity in the pH range between 4.0 and 8.0 with its pH optimum at 5.5. Several metal cations had partial inhibitory effects toward POIII. Fe³⁺ enhanced the activity of the enzyme by 160% at 5 mM. All the metal chelators caused partial inhibitory effects toward POIII, except for EDTA at 1 mM, which had no effect on the enzyme.     

Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit (Sclerocarya birrea subsp. Caffra)

Marula fruit, native to sub-Saharan Africa, is of growing commercially importance. Polyphenol oxidase (PPO) and peroxidase from the fruit were partially purified by a combination of temperature induced phase separation in Triton X-114, DEAE-ion exchange and Sephadex G100 gel filtration. PPO activity was purified 58-fold with 75% recovery while the purification factor for peroxidase was 19% with 25% recovery. The enzymes were characterised for enzyme concentration–reaction rate relationship, thermal stability, pH activity and stability, molecular weight, isoelectric point (pI) and kinetic parameters. PPO and peroxidase shared the same molecular weight (71 kDa) and pI (5.43). Thermal deactivation curves were bi-phasic for both activities. Peroxidase displayed maximal activity at pH 4.0 with ABTS (2,2′-azino-(bis-3-ethylbenzthiazoline-6-sulfonic acid)) and a K_M of 1.77 mM for hydrogen peroxide. The pH optimum for PPO was 7.0 with catechol. Marula PPO had K_M values of 1.41, 1.43, 3.73 and 4.99 mM for catechin, 4-methylcatechol, 3,4-dihydroxyphenylpropanoic acid (DHPPA) and catechol, respectively.     

ACEROLA's CLONES OF INDUSTRIAL INTEREST

In order to know which clone of acerola is better for acerola industrialization, we studied the pectin methylesterase (PME) specific activity, pectin content and vitamin C content in five different clones of acerola. The pectin yield varied from 1.37 to 2.99% and the highest content of pectin occurred in clones 3 and 5. Ascorbic acid varied significantly from 1157.5 to 1735.5 mg/100 g of pulp in the five clones. The highest content of vitamin C occurred in clone 4. The PME specific activity varied from 0.79 to 2.92 units g ^?1 /g of pulp and the highest values occurred in clone 2. We also studied the optimum temperature and the optimum pH of this enzyme. Clones 1, 2, 4 and 5 showed optimum temperature at 90C. Clone 3 showed practically the same specific activity at all temperatures studied. Clones 1 and 4 showed an optimum pH of 9.0 and clone numbers 2, 3 and 5 showed a pH optimum at 8.5.     

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp. 7-12

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ‐CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β‐CD, while from alkalophilic Bacillus sp. 7‐12. γ‐CD yield is close to β‐CD. A CGTase from alkalophilic Bacillus sp. 7‐12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and Sepharose CL‐6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS‐PAGE. The enzyme showed a K_mof 1.24 mg/mL and V_max0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag⁺, Cu²⁺, Mg²⁺, Al³⁺, Co²⁺, Zn²⁺, Fe²⁺and slightly inhibited by Sn²⁺, Mn²⁺. The ions Ca²⁺and K⁺, EDTA and DTT had no influence on the enzyme activity.