Purification and characteration of xylanase from Aspegillus oryzae VTCC F187

Xylanase is produced by many bacteria and fungi, among which Aspergillus oryzae is considered as a potential source. In this study, a xylanase was isolated and purified from the crude culture filtrate of Aspergillus oryzae VTCC F187 after 7 days of growth on the optimal culture containing 7% corncob and 5% soybean powder under liquid-state fermentation. After two steps purification process including gel filtration chromatography (Sephadex G-75) incorporating with anion-exchange chromatography (DEAE-sephadex), obtained xylanase was purified with the yield and purity of 24.9% and 3.91 fold, respectively. The molecular mass of the purified xylanase determined by SDS–PAGEwas 32 kDa with a specific activity of 1268.0 U/mg towards 1% (w/v) of birch wood xylan. The xylanase displayed its optimum activity at 60°C, pH 6.5, and the enzyme remained active effectively within pH 3.0–5.0 and at the temperature below 37°C. Some substances were tested at concentration of 2% (v/v) such as β-mercaptoethanol, DMSO, Tween 80 and 10 mM NaN3 slightly decreased xylanase activity and reached over 85%. While EDTA 10 mM and SDS at concentration of 2% inhibited more strongly, xylanase activity was only 77.6% and 56.6% comparing with control one, respectively. The biochemical characteristics suggested that the xylanase has a potential application, including use as a feed enzyme or using hydrolysis to produce environmentally friendly Bio-products.

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Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

Biochemical Journal ◽

10.1042/bj2520865 ◽

1988 ◽

Vol 252 (3) ◽

pp. 865-874 ◽

Cited By ~ 16

Author(s):

R A Harrison

Keyword(s):

Ionic Strength ◽

Quantitative Determination ◽

Specific Activity ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

High Yield ◽

Poly Vinyl Alcohol ◽

The Media ◽

Low Ionic Strength

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

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Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.1980-1987.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 1980-1987 ◽

Cited By ~ 34

Author(s):

Yolanda Sanz ◽

Fidel Toldrá

Keyword(s):

Specific Activity ◽

Divalent Cations ◽

Gel Filtration ◽

Sulfhydryl Group ◽

Fold Increase ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Lactobacillus Sakei ◽

C Terminus ◽

Arginine Aminopeptidase

ABSTRACT An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

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β-Carotene-15,15′-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure

British Journal Of Nutrition ◽

10.1079/bjn19940042 ◽

1994 ◽

Vol 72 (3) ◽

pp. 397-414 ◽

Cited By ~ 47

Author(s):

Jannine Devery ◽

B. V. Milborrow

Keyword(s):

Guinea Pig ◽

Intestinal Mucosa ◽

Specific Activity ◽

Gel Filtration ◽

Tween 80 ◽

Reversed Phase ◽

Exchange Chromatography ◽

Protein Aggregate ◽

Β Carotene ◽

Dioxygenase Activity

βCarotene-15,15′-dioxygenase (EC 1.13.11.21; β-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15′-14C2l- or [15,15′-3H2]β-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled β-carotene and β-carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining β-carotene and retinaldehyde, which was the only cleavage product detected. The labelled material produced by the enzyme was analysed by either normal-phase TLC or reversed-phase HPLC and characterized chemically as retinaldehyde. The lack of other labelled up-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol β-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More β-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The β-carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formedlmg protein per h. The formation of a lipid-protein aggregate containing the β-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal serapings were homogenized in buffer at a proportion of 1:4 (w/v).

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Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

Jurnal Agritech ◽

10.22146/agritech.17004 ◽

2017 ◽

Vol 37 (1) ◽

pp. 31

Author(s):

Fitria Fitria ◽

Nanik Rahmani ◽

Sri Pujiyanto ◽

Budi Raharjo ◽

Yopi Yopi

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Centrifugal Filter ◽

Sds Page ◽

Enzyme Specific Activity ◽

Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the xylooligosaccharide types from xylan hydrolysis by this enzyme. Based on this research, the optimum time for enzyme production occurred at 96 hours with the enzyme activity of 6.275 U/mL and enzyme specific activity of 5.093 U/mg. The specific activities were obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0 U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram showed that the molecular weight of xylanase protein were about 25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji hidrolisis untuk mengetahui jenis xilooligosakarida yang dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96 dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil pemekatan dengan amicon® ultra-15 centrifugal filter devices, kromatografi filtrasi gel dan kromatografi penukar anion mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

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Purification and characterization of an endoxylanase from Trichoderma koningii G-39

Biochemical Journal ◽

10.1042/bj2780329 ◽

1991 ◽

Vol 278 (2) ◽

pp. 329-333 ◽

Cited By ~ 31

Author(s):

L Huang ◽

T H Hseu ◽

T T Wey

Keyword(s):

Specific Activity ◽

Xylanase Activity ◽

Gel Filtration ◽

Exchange Chromatography ◽

Crystalline Cellulose ◽

Trichoderma Koningii ◽

Sds Page ◽

Immunological Relationship ◽

Oat Spelt

Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

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Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

Biochemical Journal ◽

10.1042/bj2950463 ◽

1993 ◽

Vol 295 (2) ◽

pp. 463-469 ◽

Cited By ~ 17

Author(s):

S A Freeman ◽

K Peek ◽

M Prescott ◽

R Daniel

Keyword(s):

Specific Activity ◽

Sequence Similarity ◽

Substrate Inhibition ◽

Gel Filtration ◽

Serine Proteinase ◽

Fold Increase ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Ph Optimum ◽

High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

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Cloning, Expression, and Characterization of Xylanase G2 from Aspergillus oryzae VTCC-F187 in Aspergillus niger VTCC-F017

BioMed Research International ◽

10.1155/2021/8840038 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Do Thi Tuyen ◽

Nguyen Tien Cuong ◽

Nguyen Sy le Thanh ◽

Nguyen Thi Thao ◽

Le Thanh Hoang ◽

...

Keyword(s):

Aspergillus Niger ◽

Aspergillus Oryzae ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Xylanase Activity ◽

Tween 80 ◽

Polymerase Chain Reaction Method ◽

Tween 20 ◽

Highly Active

The study focuses on engineering of recombinant Aspergillus niger to produce highly active xylanase. The xylanase G2 encoding gene originating from Aspergillus oryzae VTCC-F187 was cloned, amplified, and inserted into the pAN7.1GluA vector with specific primers possessing BamHI. The recombinant plasmid was introduced into Aspergillus niger VTCC-F017 by chemical methods. The recombinant strain was checked by polymerase chain reaction method and Southern blot. Next, the recombinant protein was expressed and purified by His-tag column. The molecular mass of the purified xylanase G2, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was 21 kDa with a specific activity of 1025 IU/mg towards 0.5% (w/v) of birchwood xylan. The optimal temperature and pH were 55°C and pH 6.5, respectively. The enzyme was stable in a temperature ranges 25–40°C and a pH ranges 5–7. The presence of Tween 80 enhanced xylanase activity. Triton X-100, however, had no impact on the function of the enzyme. The xylanase activity was reduced by Tween 20, SDS, and organic solvents. The enzyme was completely inhibited by Hg2+ and partially by Zn2+, Fe2+, and Ag+, while it was slightly stimulated by K+ and EDTA.

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Purified Factor XII Has a Higher Specific Activity than the Parent Molecule in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647478 ◽

1991 ◽

Vol 65 (02) ◽

pp. 169-173 ◽

Cited By ~ 3

Author(s):

Walter A Wuillemin ◽

Miha Furlan ◽

Isabella Huber ◽

Bernhard Lämmle

Keyword(s):

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Factor Xii ◽

Parent Molecule ◽

Proteolytic Activation ◽

Filtration Step

SummaryThe specific clot promoting activity of factor XII (F XII) in plasma samples from 50 healthy adults was between 30 and 48 U/ mg, whereas the specific activity of purified F XII ranged from 55 to 66 U/mg. This difference was neither due to partial proteolytic activation during purification of F XII nor to the influence of plasma protease inhibitors. Purified F XII showed normal size and charge, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, respectively. The increase of the specific F XII activity during the purification process mainly occurred after anion exchange chromatography on DEAE-Sephadex and after the final gel filtration step. Upon dextran sulfate activation, proteolytic cleavage of F XII and generation of kallikrein-like amidolytic activity was faster in F XII deficient plasma containing purified F XII than in F XII deficient plasma containing a corresponding amount of pooled normal plasma (NHP). The binding to kaolin was similar for both, purified F XII and plasma F XII.In conclusion, purification alters the properties of F XII in an unknown way, resulting in an increased specific clot promoting activity.

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Purification and evaluation for effects of temperature on extracellular xylanase activity from Aspergillus oryzae DSM 1863

Journal of Vietnamese Environment ◽

10.13141/jve.vol8.no1.pp9-13 ◽

2017 ◽

Vol 8 (1) ◽

pp. 9-13

Author(s):

Thi Tuyen Do ◽

Sy Le Thanh Nguyen ◽

Thi Thao Nguyen

Keyword(s):

Aspergillus Oryzae ◽

Specific Activity ◽

Xylanase Activity ◽

Residual Activity ◽

Deae Cellulose ◽

Exchange Chromatography ◽

Birch Wood ◽

Sds Page ◽

Birch Wood Xylan ◽

Effects Of Temperature

Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6-775.9 U/mg protein). Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.

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Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

Canadian Journal of Microbiology ◽

10.1139/w05-158 ◽

2006 ◽

Vol 52 (6) ◽

pp. 519-524 ◽

Cited By ~ 19

Author(s):

H N Bhatti ◽

M Madeeha ◽

M Asgher ◽

N Batool

Keyword(s):

Aspergillus Niger ◽

Glucose Oxidase ◽

Specific Activity ◽

Gel Filtration ◽

High Specificity ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Analytical Reagent ◽

Intracellular Glucose

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg–1 through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a Km value of 25 mmol L–1. The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 °C. The enzyme displayed a high thermostability having a half-life (t1/2) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol–1, and free energy of denaturation (G*) of 103.63 kJ mol–1. These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.Key words: glucose oxidase, Aspergillus niger, kinetics, thermodynamics, thermal stability.

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