Purification and Properties of the Raw Starch Digesting Amylase from Penicillium brunneum No. 24

Raw sago starch digesting amylase was obtained from Penicillium brunneum No. 24. The crude enzyme from this strain contains carboxymethylcellulase (CMC-ase), avicelase, α-amylase and α-glucosidase. Affinity chromatography (α-cyclodextrin-Sepharose 6B) of the enzyme after ammonium sulfate fractionation, Toyopearl HW-55F gel filtration, DEAE-Sephadex A-50 and DEAE-cellulose chromatographies fractionation steps, resulted in a homogeneous glucoamylase. SDS-polyacrylamide gel electrophoresis of purified enzyme showed a single band, and a molecular weight of 80,000 for the native glucoamylase from Penicillium brunneum No. 24 was observed. After modification of the native glucoamylase with subtilisin, the molecular weight was reduced to 76,000. It lost the ability to digest and adsorb onto raw starches. However, its ability to digest gelatinized starches was preserved.     

Polyphenol Oxidase from Bean Sprouts (Glycine max L.)

ABSTRACT: Polyphenol oxidase (PPO) was purified and characterized from bean sprouts by ammonium sulfate precipitation, DEAE‐Toyopearl 650M, CM‐Toyopearl 650M, SuperQ‐Toyopearl 650S and QAE‐Toyopearl 550C column chromatographies. Substrate staining of the crude extract on electrophoresis showed the presence of 2 isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be about 54 kDa. The optimum pH was 9.0 and optimum temperature 40 °C. Heat inactivation occurred about 30 °C. PPO showed activity to catechol, pyrogallol and dopamine. These compounds such as ascorbic acid, L‐cysteine, 2‐mercaptoethanol, and glutathione used was the effective inhibitor. Enzyme activity was maintained for 7 d at 4 °C but suddenly decreased after 8 d.     

Transglutaminase from Streptoverticillium ladakanum and application to minced fish product

The transglutaminase (TGase) from Streptoverticillium ladakanum was purified to electrophoretic homogeneity after ammonium sulfate fractionation and Blue Sepharose Fast Flow chromatography. The molecular weight of the purified TGase was 30.5 kDa estimated by Superdex 75HR gel filtration, and 37.5 kDa by SDS-PAGE. This enzyme, with optima at pH at 6.0 and 50°C was very stable at pH 5.0–7.0. It was strongly inhibited by PCMB, PMSF, Pb²⁺, Zn²⁺ and Cu²⁺, but not affected by EDTA and Ca²⁺. This suggested that the purified TGase was calcium-independent and its active center contained cysteine. It catalyzed the crosslinking of fish myosin heavy chain and substantially increased the gel strength of mackerel surimi.     

A Novel Type Mitochondrial Monoamine Oxidase from the Liver of Skipjack Tuna (Katsuwonus pelamis): Purification and Characterisation

A novel type of monoamine oxidase (EC. 1.4.3.4) present in the liver of skipjack tuna (Katsuwonus pelamis) was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by ammonium sulphate fractionation, followed by Sephadex G-200, butyl-toyopearl 650 M, phenyl-toyopearl 650 M and hydroxyapatite chromatography. The molecular weight of the enzyme was estimated to be about 110000 by gel filtration on Sephadex G-200. The enzyme was activated by Mn²⁺, but was inhibited by Cu²⁺, Zn²⁺ and p-chloromercuribenzoic acid (PCMB). Furthermore, the enzyme was completely inhibited by Hg²⁺ and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). © 1997 SCI.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

PURIFICATION AND PROPERTIES OF TRANSGLUTAMINASE FROM STREPTOVERTICILLIUM MOBARAENSE

Transglutaminase (TGase) was separated from the culture broth of an isolated strain of Streptoverticillium mobaraense. The crude enzyme was prepared by centrifugation, ultrafiltration, precipitation by alcohol, centrifugation and freeze‐drying. The yield after these processes was 65–70%. Then the enzyme was purified to homogeneity by chromatography on CM‐cellulose and Sephadex G‐75 on which the yields were about 70% and 80%, respectively; the purified folds reached 2.5–4.7 and 1.08–2.06, respectively. The molecular weight of this TGase was 39,500–40,100 Da by gel filtration chromatography. Optimum enzyme activity was observed in the pH range of 5.0–7.0, and it was maintained stable at 20–40C. The optimal temperature and pH was 52C and 6.0, respectively. At 1 mM and 5 mM metal ion or inhibitors concentration, TGase activity was strongly inhibited by Zn²⁺ and NEM, and not affected obviously by Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Na⁺ as well as PMSF and EDTA. The effects of these additions on this TGase were compared with those of other microbial TGases.     

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 Pullulanase and α-Amylase Activities of a Thermus sp. AMD33

Thermostable Thermus sp. AMD 33 pullulanases (I and II) capable of cleaving α-1,6-links in pullulan as well as α-1,4-glucosidic linkages in amylose were purified to electrophoretically homogeneous states. Relative molecular masses and pI values were determined as 135,000 (I and II) by SDS-PAGE and 4.2 (I) and 4.3 (II) by isoelectric focusing, respectively. The pullulanase and α-amylase activities of the purified enzyme II responded similarly to temperature and pH, with optima at 70°C and pH 5.5–6.0. Both activities were activated by Ca²⁺ and inhibited by Hg²⁺, Fe³⁺, NBS, DBS, SDS and urea to almost the same extent. Both activities were also inhibited competitively by CDs. Enzyme II catalyzed the hydrolysis of α-1,6-glucosidic linkages in maltosyl- and maltotriosyl-α-CD as well as that of α-1,4-bonds in amylose and related linear malto-oligosaccarides larger than maltotriose, but exhibited no action on panose, isopanose or glucosyl α-CD.     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

Purification and characteristics of serine protease from the head of pacific white shrimp

Serine protease from the head of Pacific white shrimp was purified by the following techniques: ammonium sulfate fractionation, Q-Sepharose HP ion exchange chromatography, and Sephadex G-100 gel filtration. The molecular weight was estimated as 32.8 kDa using SDSPAGE. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 10.0 and 40°C. It was stable at pH range from 8.0 to 11.0 and had good thermal stability. Pb²⁺, Ca²⁺, Mg²⁺, Cu²⁺, and Mn²⁺ could active the enzyme certainly when Zn²⁺ and Hg²⁺ strongly inhibited the activity. The enzyme was inhibited by the general serine protease inhibitor (PMSF) and the specific trypsin inhibitors (TLCK, SBTI). The modification of various amino acid modifiers for the purified enzyme determined that the enzyme active center included tryptophan, histidine, and serine, moreover, arginine had a certain relationship with the enzyme activity.     

A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

Microbial demetallization of crude oil: nickel protoporphyrin disodium as a model organo-metallic substrate

A soil isolate designated as YA-1 strain was selected for its ability to degrade nickel protoporphyrin disodium (NiPPDS). The strain was capable of utilizing NiPPDS as the sole source of carbon. This strain, a gram-negative aerobic rod, was identified as Pseudomonas azelaica YA-1 based on the result of its 16S rRNA analysis. Product analyses by HPLC showed that this strain can decompose the porphyrin ring to which a metal ion is bound. However, the use of whole bacterial cells cannot result in extensive NiPPDS degradation; therefore, the YA-1 enzyme was extracted and purified. This NiPPDS-degrading enzyme named as protoporphyrinase was purified from P. azelaica YA-1 by ammonium sulfate fractionation and sequential chromatographies using DEAE Toyopearl 650 M, CM Toyopearl 650 M and Biogel P-60 columns, with a yield of 11.3% based on the enzyme activity and an overall purification of 498-fold. The molecular weight of this enzyme is estimated to be 39,000 Da by SDS-PAGE and 34,000 Da by gel filtration. The optimum pH and temperature for the enzyme were 7.0 and 30 degrees C, respectively. The activity was stable at pH 2.0-11.0 and at temperatures below 50 degrees C. The enzyme activity was inactivated by ferric chloride, potassium ferricyanide, ZnCl2 and CdCl2.     

Purification and Properties of Beta-galactosidase from Streptococcus thermophilus

A β-galactosidase from Streptococcus thermophilus was purified to homogeneity by ammonium sulfate and acetone fractionation, gel filtration on Sephadex G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme preparation exhibited an optimum pH at 6.6–7.0 and an optimum temperature of 57°C. The enzyme was stable at pH 6.8–7.0. Km and V_max for the enzyme, using ortho-nitrophenyl β-D-galactopyranoside as the substrate, were 0.25 mM and 83 μmoles/mg protein/min, respectively. It was strongly inhibited by Hg⁺⁺, Ag⁺, and Cu⁺⁺ as well as pchloro-mercuri benzoate. The enzyme had a molecular weight of about 6 × 10⁵ and was highly specific for β-galactoside bonds.     

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.     

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.     

Purification and Characterization of a Thermophilic α-Amylase of Aspergillus niger van Tieghem

The exocellular α-amylase of a strain of Aspergillus niger van Tieghem (elaborating both dextrifying and saccharifying thermophilic amylases) was purified to homogeneity. Purification was achieved by providing cultural conditions for the organism to preferentially synthesize α-amylase and fractionation of the culture filtrate by DEAE-Sephadex chromatography. Its purity was established by gel electrophoresis and confirmed by sedimentation studies. The molecular weight of the enzyme was 56,230. Its Km values on different starches, temperature and pH optima for activity and energy of activation were established. Compared to literature values for other fungal α-amylases, this enzyme exhibited a lower energy of activation, increased tolerance to lower pH and enhanced affinity to starch, highlighting its potential industrial application. While Ag⁺, Pb²⁺, Hg⁺, Al³⁺ and EDTA inhibited the activity of the enzyme, Ca²⁺ enhanced its activity, apart from conferring thermal stability and lowered activation energy. The product of its action on starch were maltooligosaccharides, maltose and glucose.     

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     

Purification and Properties of Inulinase from Arthrobacter globiformis S64—1

A bacterium, Arthrobacter globiformis S64—1, produced an inulinase in the culture broth. The enzyme was purified 442-fold by DEAE-Toyopearl chromatographies. It showed maximal activity at 40°C and pH 6.5. The enzyme activity was inhibited strongly by Hg²⁺, Fe³⁺, Cu²⁺ and EDTA. The molecular weight of the enzyme was estimated to be 100,000 by SDS-PAGE. The isoelctric point of the enzyme was estimated to be 4.8 by isoelectric focusing on a polyacrylamide gel. The enzyme degraded inulin through an exo-type reaction.     

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 properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.