Purification and characterization of the extracellular amylases of the yeast Schwanniomyces alluvius

1984 ◽  
Vol 30 (9) ◽  
pp. 1163-1170 ◽  
Author(s):  
B. Simões-Mendes
Keyword(s):  
Deae Cellulose ◽  
Culture Fluid ◽  
Soluble Starch ◽  
Ph Stability ◽  
Starch Hydrolysis ◽  
Heat Inactivation ◽  
Optimum Temperature ◽  
Enzyme Preparations ◽  
Optimum Ph

The extracellular amylolytic system of a strain of the yeast Schwanniomyces alluvius consists of an α-amylase, a glucoamylase, and probably a debranching enzyme. Crude enzyme preparations were obtained by fractionation of the culture fluid, at the stationary phase of growth, with isopropanol. Purification was carried out by DEAE-cellulose chromatography. The glucoamylase had the following properties: molecular weight (MW), 117 000 ± 2300; optimum temperature, 50 °C; optimum pH, 4.5; range of pH stability, pH 4–6; final product of starch hydrolysis, glucose; ΔH≠ and ΔS≠ of heat inactivation, 39747 cal∙mol−1 and 274.3 cal∙deg−1∙mo−1; Km(30 °C, pH 4.5) for soluble starch, 22.22 g∙L−1. The α-amylase had the following properties: MW, 62 000 ± 500; optimum temperature, 40 °C; optimum pH, 6.3; range of pH stability, pH 4–7; final product of starch hydrolysis, maltose and glucose; ΔH≠ and ΔS≠ of heat inactivation, 36594 cal∙mol−1 and 256.9 cal∙deg−1 mol−1; Km (40 °C, pH 5.5), 2.7 g∙L−1.

Immobilization and Characterization of Tannase and its Haze-removing

2009 ◽  
Vol 15 (6) ◽  
pp. 545-552 ◽  
Author(s):  
Erzheng Su ◽  
Tao Xia ◽  
Liping Gao ◽  
Qianying Dai ◽  
Zhengzhu Zhang
Keyword(s):  
Kinetic Parameter ◽  
High Activity ◽  
Green Tea ◽  
Storage Stability ◽  
Residual Activity ◽  
Black Tea ◽  
Optimum Temperature ◽  
Oolong Tea ◽  
Optimum Ph

Tannase was effectively immobilized on alginate by the method of crosslinking-entrapment-crosslinking with a high activity recovery of 76.6%. The properties of immobilized tannase were investigated. Its optimum temperature was determined to be 35 ° C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH of 5.0 did not change. The thermal and pH stabilities of immobilized tannase increased to some degree. The kinetic parameter, Km, for immobilized tannase was estimated to be 11.6 × 10-4 mol/L. Fe2+ and Mn2+ could activate the activity of immobilized tannase. The immobilized tannase was also applied to treat the tea beverage to investigate its haze-removing effect. The content of non-estern catechins in green tea, black tea and oolong tea increased by 52.17%, 12.94% and 8.83%, respectively. The content of estern catechins in green tea, oolong tea and black tea decreased by 20.0%, 16.68% and 5.04%, respectively. The anti-sediment effect of green tea infusion treated with immobilized tannase was significantly increased. The storage stability and reusability of the immobilized tannase were improved greatly, with 72.5% activity retention after stored for 42 days and 86.9% residual activity after repeatedly used for 30 times.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

A fundamental thermostable cyanide-sensitive phosphoenolpyruvate acid carboxylase in photosynthetic and other organisms

1971 ◽  
Vol 49 (4) ◽  
pp. 631-643 ◽  
Author(s):  
David Pan ◽  
E. Roy Waygood
Keyword(s):  
Heat Treatment ◽  
Zea Mays ◽  
Acid Phosphatase ◽  
Plant Species ◽  
Phosphoenolpyruvate Carboxylase ◽  
Deae Cellulose ◽  
Bundle Sheath ◽  
Evolutionary Significance ◽  
Optimum Temperature ◽  
Optimum Ph

A thermostable 'phosphoenolpyruvate carboxylase' has been isolated from leaves of Zea mays different from phosphoenolpyruvate carboxylase (EC. 4.1.1.31) in that its optimum pH is 5.4, it does not liberate orthophosphate during the reaction, and it is inhibited by cyanide. The enzymic reaction has an optimum temperature of 70–75C and has been purified through steps including acidification to pH 4.6, heat treatment to 50C, and DEAE-cellulose and Sephadex G-200 column chromatography. Three fractions were active in the Sephadex eluate, but only fraction III was free from a thermostable acid phosphatase which catalyzes the liberation of orthophosphate from the substrate and the end product which is suggested to be a C4 phosphocarbonyl compound, although phosphohydroxypyruvate appears by either spontaneous or enzymic decarboxylation. The enzyme is assayed by the formation of a phenyl-hydrazone at 325 nm. The enzyme is localized and tightly bound in both the parenchyma bundle sheath and mesophyll chloroplasts, which are free from the thermostable acid phosphatase. Similar concentrations of the enzyme have been found in all plant species tested including C3 plants, ferns, bryophytes, algae, fungi, and even in calf liver. The enzyme must have considerable evolutionary significance.

scholarly journals The characterization of two reduced nicotinamide–adenine dinucleotide phosphate-linked aldehyde reductases from pig brain

1972 ◽  
Vol 130 (3) ◽  
pp. 765-772 ◽  
Author(s):  
A. J. Turner ◽  
K. F. Tipton
Keyword(s):  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Aldehyde Reductase ◽  
Enzyme Preparations ◽  
Pig Brain ◽  
Michaelis Constants ◽  
Probable Role ◽  
Reduced Nicotinamide Adenine Dinucleotide

1. NADPH-linked aldehyde reductase from pig, ox and rat brain exhibits non-linear reciprocal plots when partially purified enzyme preparations are studied. 2. In pig brain this non-linearity is due to the presence of two distinct aldehyde reductases, which can be separated by DEAE-cellulose chromatography. 3. These two enzymes can be distinguished by several criteria, including pH optima, Michaelis constants for substrates and their inhibitor sensitivity. 4. The probable role of these enzymes in the metabolism of the aldehydes derived from the biogenic amines is discussed.

scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

scholarly journals Purification and characterization of endoglucanase Ss from Clostridium thermocellum

1991 ◽  
Vol 279 (1) ◽  
pp. 67-73 ◽  
Author(s):  
U Fauth ◽  
M P M Romaniec ◽  
T Kobayashi ◽  
A L Demain
Keyword(s):  
Clostridium Thermocellum ◽  
Hydrophobic Interaction Chromatography ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Specific Antibodies ◽  
Optimum Ph ◽  
Exchange Adsorption

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate known as the cellulosome. By using a combination of ion-exchange, adsorption and hydrophobic-interaction chromatography, it was possible to isolate from extracellular broth a specific endoglucanase of interest without the use of denaturants. The endoglucanase was identified as the cellulosomal subunit Ss by the use of specific antibodies. The enzyme has an Mr of 83,000, an isoelectric point of 3.55, optimum pH of 6.6 and optimum temperature of 70 degrees C. It hydrolyses CM-cellulose and, at a higher rate, the cellodextrins, cellotetraose and cellopentaose, but does not hydrolyse a crystalline cellulose such as Avicel. Cellobiose and cellotriose are also immune to attack. It differs from endoglucanases previously isolated by others and a 76,000-Mr endoglucanase recently isolated in this laboratory.

PURIFICATION AND PARTIAL CHARACTERIZATION OF THERMOACTINOMYCES VULGARIS AMYLASES

1967 ◽  
Vol 13 (9) ◽  
pp. 1157-1163 ◽  
Author(s):  
M. J. Kuo ◽  
P. A. Hartman
Keyword(s):  
Ion Exchange ◽  
Column Chromatography ◽  
Maximum Rate ◽  
Amylase Activity ◽  
Enzyme Preparations ◽  
Ph Values ◽  
Ion Exchange Column ◽  
Optimum Ph ◽  
Thermoactinomyces Vulgaris

An α-amylase from Thermoactinomyces vulgaris has been purified about 100-fold. Its optimum pH was between 5.9 and 7.0, and the maximum rate was achieved at 60 °C. In the absence of substrate, the enzymes were more stable at pH 5.9 than at higher or lower pH values; inactivation was rapid at pH 7.0. Temperatures of 70 °C or greater also caused rapid denaturation of the enzyme in the absence of substrate. Three major peaks of amylase activity were detected when purified enzyme preparations were passed through Sephadex G-75 columns. At least two of these amylases were interconvertible. Four or five T. vulgaris proteinases also were separated, using ion exchange column chromatography.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

scholarly journals The Properties of Immobilized Invertase Onto a New Support Material; Poly(Methacrylamide/Maleic Acid) Copolymeric Hydrogel

2018 ◽  
Vol 26 (2) ◽  
pp. 307-328 ◽  
Author(s):  
Hesna Nursevin Öztop ◽  
Fatma Banu Çatmaz ◽  
Dursun Saraydin
Keyword(s):  
Maleic Acid ◽  
Storage Stability ◽  
Maximum Velocity ◽  
Optimum Temperature ◽  
Michaelis Constant ◽  
Ph Values ◽  
Optimum Ph ◽  
And Storage ◽  
And Diffusion

Abstract Poly (methacrylamide / maleic acid) PM/MA and poly (methacrylamide) PM hydrogels were prepared aiming to be used as a support for invertase. Spectrophotometric, thermal analysis methods, swelling and diffusion experiments were used for the characterization of hydrogels. The swelling of PM/MA was higher than that of PM in water. The diffusion of water within the hydrogel was found to be non-Fickian. Invertase was immobilized onto PM and PM/MA (samples named PM-I and PM/MA-I respectively). The optimum pH values were found to be; 6.0, 5.0 and 5.5 for free invertase, PM-I and PM/MA-I respectively. The optimum temperature values were found to be 30 °C, 35 °C and 40 °C for free invertase, PM-I and PM/MA-I respectively. The Michaelis constant (Km) and maximum velocity of the enzymes (Vmax) were Km: 11,75 mM, Vmax: 1,95 μmol min−1 for free invertase, Km: 67,24 mM, Vmax: 60,6 μmol min−1 for PM-I and Km: 74,55 mM, Vmax: 18,12 μmol min−1 for PM/MA-I. PM/MA-I showed excellent thermal, operational and storage stability.

scholarly journals Amylolytic ability of bacteria isolated from termite (Coptotermes sp.) gut

2018 ◽  
Vol 23 (1) ◽  
pp. 14 ◽  
Author(s):  
Putri Dwi Mulyani ◽  
Radhiyah Mardhiyah Hamid ◽  
Rifqi Zahroh Janatunaim ◽  
Yekti Asih Purwestri
Keyword(s):  
Amylase Activity ◽  
Rrna Gene ◽  
Optimum Temperature ◽  
Bacterial Isolates ◽  
16S Rrna Gene Sequences ◽  
Clear Zone ◽  
Optimum Ph ◽  
Iodine Test ◽  
Brevibacillus Parabrevis

BSR 2, BSR 3, BSR 8, and BSR 9, different bacteria isolated from the termite gut, have been shown to possess cellulolytic activities, but their amylolytic ability has heretofore been unknown. This study attempted to fill in this knowledge gap. The formation of a clear zone using the iodine test showed that the bacteria were able to produce and secrete amylase. Based on the results, the best cultivation times for strains BSR 2, BSR 3, BSR 8, and BSR 9 were 6, 3, 2, and 2 d, respectively, yielding amylase activities of 2.59 ± 0.13 U/mg, 2.00 ± 0.08 U/mg, 1.67 ± 0.10 U/mg, and 1.55 ± 0.12 U/mg, respectively. BSR 2 had the highest amylase activity compared with the other bacterial isolates. The optimum ph for bacterial amylase activity of BSR 2 was 7.0, and the optimum temperature was 40°C. The molecular characterization of isolates BSR 2, BSR 3, BSR 8, and BSR 9 was based on 16S rRNA gene sequences. Isolates BSR 8 and BSR 9 were thus identified as Brevibacillus parabrevis and Brevibacillus sp. With similarities amounting to 92.48% and 95.91%, while the BSR 3 isolate was identified as Pseudomonas alcaligenes with a similarity of 94.29%, and the BSR 2 isolate could not be identified yet.

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