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.     

Purification and characterization of cathepsin B from the gut of the sea cucumber (<Emphasis Type="Italic">Stichopus japonicas</Emphasis>)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu²⁺, Ni²⁺, and Zn²⁺ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.     

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.     

Purification and Characterization of a β-Amylase of Hendersonula toruloidea

β-Amylase produced by Hendersonula toruloidea was purified to homogeneity by salting out with ammonium sulphate, ion-exchange chromatography on DEAE-cellulose and gel-filtration on Sephadex G-75. The relative molecular mass of the enzyme was estimated to be 60,000 by gel filtration. The enzyme was optimally active at pH 6.0 and 60°C, stable between pH 6 and 8 (24 h) and retained 74% activity at 70°C (30 min). It was strongly activated by Na⁺ but inhibited by Hg²⁺, Zn²⁺ and Cu²⁺. The enzyme hydrolyzed amylopectin (Km 0.42 mg/ml) forming maltose, maltotetraose and unidentified maltooligosaccharide, and hydrolyzed soluble starch (Km 0.3 mg/ml) and glycogen (Km 0.5 mg/ml) forming maltose and unidentified maltooligosaccharide.     

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0?7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca ²⁺ , Mn²⁺, PCMB, and DTNB, but inhibited by Ba²⁺, Fe³⁺, Hg²⁺, Mg²⁺, and iodoacetic acid. The purified enzyme was of α?-type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α?-1,4-linkages and with increasing percentage of α?-1,6-linkages in substrate similar to the results from commercially available honey.     

INHIBITION OF SHIKIMATE DEHYDROGENASE FROM HEART-OF-PALM (EUTERPE OLERACEA MART.)

Shikimate dehydrogenase (E.C.1.1.1.25), extracted from heart-of-palm, was purified by precipitation with ammonium sulphate and elution from columns of Sephadex G-25, and DEAE-Sephadex A-50. The enzyme was unstable and it was not possible to maintain the activity by the addition of cysteine and 2-mercaptoethanol to the buffer. The heart-of-palm contained two isoenzymes which were separated by polyacrylamide gel electrophoresis. The enzyme was inhibited by metal ions and p-CMB. The p-CMB inhibition was reversed by cysteine. The shikimate dehydrogenase was competitively inhibited by protocatechuic acid (Ki 7.5 × 10^?4M), and Km values of 0.021 mM and 0.011 mM for shikimic acid and NADP⁺, respectively, were obtained at pH 9.5 in Tris-HCl-glycine buffer. The molecular weight determination was performed on Sephadex G-100 and the MW was estimated to be 50,000 daltons.     

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 Properties of β-Glucuronidase from the Muscle of Bombay Duck (Harpodon Nehereus)

Bombay duck muscle β-glucuronidase purified 4200-fold had molecular weight of 160,000 as estimated by gel filtration on Sephadex G-200. The glycoprotein enzyme exhibited dimeric structure on SDS-PAGE and had a pI of 5.0. The enzyme was optimally active at pH 5.2 when phenolphthalein β-D-glucuronide was used as the substrate while with p-nitrophenyl glucuronide the pH optimum was 4.6. Saccharo-1,4-lactone was a potent competitive inhibitor of the enzyme. Heavy metalic ions such as Hg²⁺, Cd²⁺ and Ag⁺ also proved to be inhibitory to the enzyme. Radiation inactivation of the enzyme could be protected in the presence of mercaptoethanol. Sodium chloride activated the enzyme while sodium tripolyphosphate inhibited it.     

Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus

Transglutaminase (TGase, EC 2.3.2.13) from a Streptomyces hygroscopicus strain isolated from soil was purified from culture broth by ethanol precipitation, followed by successive chromatographies on CM-cellulose and Sephadex G-75 columns with a yield and purification-fold of 21.1% and 30%, respectively. The enzyme’s molecular weight was estimated as 38,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified microbial transglutaminase (MTG) exhibited optimum activity at 37–45 °C and in a range of pH 6.0–7.0 for hydroxamate formation from N-carboxybenzoyl-l-glutaminyl-glycine and hydroxylamine. The enzyme was not stable above 50 °C and was stable within a pH range of 5.0–8.0 at lower temperature. The MTG was not inhibited by Ca²⁺ and ethylenediaminetetraacetic acid, suggesting it was calcium-independent. Purified MTG was strongly inactivated by 5,5′-dithiobis (2-nitrobenzoic acid), Cu²⁺, Zn²⁺, Pb²⁺, and Hg²⁺, suggesting that this enzyme could possess a thiol group at the active site. The MTG stability was strongly affected by ethanol concentration. The enzyme activity was slightly elevated at a lower concentration of ethanol at 25 °C.     

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.     

Purification and characterisation of basic ascorbate oxidase from satsuma mandarin (Citrus unshiu Marc)

Basic ascorbate oxidase of the multiple enzyme forms existing in young fruit of satsuma mandarin (Citrus unshiu Marc) has been separated and subsequently purified to electrophoretic homogeneity through (NH₄)₂SO₄ fractionation and chromatographies on DEAE-Toyopearl 650M, CM-Sephadex C-50 and Sephadex G-100. The native molecular weight was estimated to be 141 kDa by gel filtration and composed of two non-identical subunits with an apparent mass of 74 kDa and 62 kDa. The optimum pH was found to be 5.5 with reasonable stability between pH 5 and 8. The enzyme had an optimum temperature at 45°C and was stable up to 50°C upon heat treatment for 5 min. The presence of sodium diethyldithiocarbamate, metabisulphite and potassium cyanide completely inhibited the enzyme activity. Fluoride also inhibited the activity substantially at higher concentrations. Other tnonovalent and divalent metal ions did not have inhibitory effects.     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry     

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

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

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

Purification and Characterization of Polyphenol Oxidase from Hemşin Apple (Malus communis L.)

The polyphenol oxidase (PPO) enzyme was purified and characterized from Hem?in Apple (Malus communis L.), which was organically grown in Hem?in, in the Rize province of Turkey. Enzyme (PPO) activation was determined with catechol substrate. Apples were homogenized with homogenate buffer (pH 8.5). This process was followed by precipitation with (20–80%) saturated solid (NH₄)₂SO₄ and dialysis. Finally, purification with DE52-Cellulose ion-exchange and Sephadex G-25 columns was performed. Experiments were performed at an optimum pH (5.5) and optimum temperature (30–40°C). The kinetic and thermal parameters Km (3.40 mM), Vmax (333.3 EU/mL.min), Ea (3.57 kcal), ?H (2.968 kcal/mol), Q₁₀ (1.33), k_cat (24.57 min^?1) and V₀ (7.2x10³ mM^?1.min^?1) were assessed. Additionally, the effects of Mg²⁺, Pb ²⁺, Fe²⁺, Fe³⁺, Cd²⁺, Cu²⁺, Zn²⁺, Co²⁺, Al³⁺, Mn²⁺ and Na⁺ on enzyme activity was recorded, and the IC₅₀ values, K_? values and inhibition types were determined.     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

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 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.     

Acid Proteinases from Antarctic Krill, Euphausia superb: Partial Purification and Some Properties

The presence of neutral and alkaline proteinases in the extract from Antarctic krill, Euphausia superba was confirmed. In addition to these enzymes, acid proteinases were found in the extract from E. superba and the two major acid proteinases, A and B, were purified 80–140 fold. The molecular weights of acid proteinases A and B were estimated by gel filtration on Sephadex G-100 to be approximately 45,000 and 64,000, respectively. The optimal pH of enzymes A and B was 3.0 toward hemoglobin. They were partially inhibited by acid proteinase specific reagents: diazoacethyl-DL-norleucine methylester (DAN) and 1,2epoxy-3-(p-nitrophenoxy) propane (EPNP). However, enzyme B was less sensitive to pepstatin while enzyme A was inhibited by pepstatin like almost all other acid proteinases.     

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