Purification and characterization of mouse pancreatic α-amylase

• 1.1. Mouse pancreatic α-amylase (EC 3.2.1.1) was purified in a two-step procedure comprising isoelectric focusing and chromatography on DEAE-Sephadex.
• 2.2. The enzyme was found to be composed of one polypeptide chain with a molecular weight of 54,000 as determined by equilibrium sedimentation and 58,000 as determined by electrophoresis in polyacrylamide gel containing sodium dodecylsulphate.
• 3.3. S°_20w was found to be 4.6 · 10⁻¹³ sec.
• 4.4. The amino acid composition is presented and is found to be remarkably similar to that of rat pancreatic α-amylase.
• 5.5. The isoelectric point was 7.0 as determined by isoelectric focusing in column.
• 6.6. The neutral sugar content was 1.5 %.
• 7.7. The specific activity was comparatively low, 211 U/mg amylase.

     

Purification and characterization of a thermostable α-amylase fromBacillus stearothermophilus

A soil isolate of Bacillus stearothermophilus was found to synthesize thermostable alpha-amylase. The enzyme was purified to homogeneity by ammonium sulfate fractionation and IECC on DEAE-cellulose column. The purified enzyme was considered to be a monomeric protein with a molar mass of 64 kDa, as determined by SDS-PAGE. The enzyme showed a wide range of pH tolerance and maximum activity at pH 7.0. The temperature tolerance was up to 100 degrees C with more than 90% catalytic activity; the maximum activity was observed at 50 degrees C. Divalent metal ions exhibited inhibitory effect on the enzyme activity. However, proteinase inhibitor did not react positively.     

Ostrich α-amylase: Purification and characterization of the pancreatic isoenzymes

• 1.1. Four ostrich pancreatic α-amylase isoenzymes were isolated by isoelectric focusing, following affinity chromatography on cyclohepta-amylose-Sepharose 4B.
• 2.2. Amino acid compositions of the four isoenzymes are very similar with only one charged amino acid (Arg) being significantly different.
• 3.3. The molecular weights, as determined by SDS-PAGE and amino acid composition, are nearly identical (52–53 kDa) for all four isoenzymes.
• 4.4. The four α-amylase isoenzymes appear to be kinetically distinct enzymes with a requirement for calcium.
• 5.5. Ostrich α-amylase isoenzymes appear to be non-glycosylated and contain one free thiol group.

     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Purification and characterization of a truncatedBacillus subtilis α-amylase produced byEscherichia coli

ABacillus subtilis amylase gene was inserted into a plasmid which transferred toEscherichia coli. During cloning, a 3 region encoding 171 carboxyterminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of theB. subtilis complete -amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with aK _m of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purified enzyme was stable at temperatures up to 50°C. While Hg²⁺, Fe³⁺ and Al³⁺ were effective in inhibiting the truncated enzyme Mn²⁺ and Co²⁺ considerably enhanced the activity.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification and characterization of β-amylase from Curculigo pilosa

Curculigo pilosa is traditionally used in the manufacture of sorghum beer in West Africa. β-Amylase was purified 100-fold with 38% yield from a crude extract, giving final specific activities of 4850 U/mg and 5650 U/mg using soluble starch and p-nitrophenyl maltopentaoside, respectively, as substrates. The molecular mass of the monomeric enzyme was 64 kDa and its pI 4.2. Both activity and thermostability are higher than reported for other plant β-amylases. The catalytic efficiency was lower for amylose than for starches and amylopectin. In contrast to other plant amylases, the β-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In this respect, it resembles β-amylases from microbial origin. This property, and its high activity and stability, explain its traditional use in the manufacture of infant food and sorghum beer in Burkina Faso and could make it applicable for other biotechnological purposes. Received: 4 March 1999 / Received revision: 6 August 1999 / Accepted: 13 August 1999     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Purification and biochemical characterization of a detergent stable α-amylase from Pseudomonas stutzeri AS22

This study reports the purification and biochemical characterization of a novel maltotetraose-forming-α-amylase from Pseudomonas stutzeri AS22, designated PSA. The P. stutzeri α-amylase (PSA) was purified from the culture supernatant to homogeneity by Sepharose mono Q anion exchange chromatography, ultrafiltration and Sephadex G-100 gel filtration, with a 37.32-fold increase in specific activity, and 31% recovery. PSA showed a molecular weight of approximately 57 kDa by SDS-PAGE. The N-terminal amino acid sequence of the first 7 amino acids was DQAGKSP. This enzyme exhibited maximum activity at pH 8.0 and 55°C, performed stably over a broad range of pH 5.0 ≈ 12.0, but rapidly lost activity above 50°C. Both potato starch and Ca²⁺ ions have a protective effect on the thermal stability of PSA. The enzyme activity was inhibited by Hg²⁺, Mn²⁺, Cd²⁺, Cu²⁺, and Co²⁺, and enhanced by Ba²⁺. PSA belonged to the EDTA-sensitive α-amylase. The purified enzyme showed high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), and oxidizing agents, such as sodium per borate and H₂O₂. In addition, PSA showed excellent compatibility with a wide range of commercial solid and liquid detergents at 30°C, suggesting potential application in the detergent industry. Maltotetraose was the specific end product obtained after hydrolysis of starch by the enzyme for an extended period of time, and was not further degraded.     

Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65–70 °C, it was rapidly deactivated at 70 °C with a half-life of 7 min and at 50 °C, the half-life was 94 min. The K _m and V _max for starch hydrolysis were 2.6 mg ml⁻¹ and 909 U mg⁻¹, respectively. Ca²⁺ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg²⁺, Ag²⁺, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2–D4) were formed more predominantly than larger maltooligosaccharides (D5–D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.     

Purification and properties of an α-amylase inhibitor from wheat

Four inhibitors of α-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-change chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60–90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Purification and characterization of α-glucosidase from Mucor rouxii

• 1.1.|α-Glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from Mucor rouxii was purified by treatment which included heat shock, precipitation with protamine sulfate, (NH₄)₂SO₄ and ethanol, and chromatography on DEAE-Sephadex and Sephadex G-200.
• 2.2.|Purity of the enzyme was assessed by gel chromatography and polyacrylamide gel electrophoresis. Only one band of protein was observed.
• 3.3.|The enzyme appeared to be a glycoprotein containing 40% carbohydrate. Glucose and, to a lesser extent, galactose and fucose were identified in acid hydrolysates of the enzyme. The enzyme behaved as a cation at acid pH but not as an anion at alkaline pH. It is suggested that possibly carboxylic groups from aspartate and/or glutamate are blocked.
• 4.4.|The optimum pH with phosphate buffer was found to be 3.5 and the optimum temperature 50–60°. The enzyme was highly specific. It failed to hydrolyse several disaccharides and synthetic α-glucosides. It hydrolysed maltose and starch only. The sole product formed from maltose was glucose. Glucose inhibited enzymatic activity, whereas glucosamine had no effect.

     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Purification and characterization of α-glucosidase in Apis cerana indica

Apis cerana indica foragers were used for the isolation of a full-length α- glucosidase cDNA, and for purification of the active nascent protein by low salt extraction of bee homogenates, ammonium sulphate precipitation and diethylaminoethyl-cellulose and Superdex 200 c hromatographies. The molecular mass of the purified protein was estimated by polyacrylamide gel electrophoresis resolution, and the pH, temperature, incubation, and substrate optima for enzymic activity were determined. Conformation of the purified enzyme as α-glucosidase was performed by BLAST software homology comparisons between matrix assisted laser desorption ionization time of flight mass spectroscopy analysed partial tryptic peptide digests of the purified protein with the predicted amino acid sequences deduced from the α-glucosidase cDNA sequence.     

Purification and properties of heat stable α-amylase from Bacillus brevis

Summary An extracellular -amylase has been isolated from a continuous culture of a thermophilic strain of Bacillus brevis. This enzyme was purified eightfold and obtained in electrophoretically homogenous form. The enzyme had a molecular weight of about 58000, a pH optimum from 5.0 to 9.0 and a temperature optimum at 80°C. The half-life of the purified enzyme in the presence of 5 mM CaCl₂ at 90° C and pH 8.0 was 20 min. The K _m value for soluble starch was calculated to be 0.8 mg/ml.     

Purification of α-amylase from Bacillus lentus cultures

Acetone fractionation of Bacillus lentus culture filtrate yielded the highest -amylase activity and the 66.6% fraction reached 13-fold that of the crude enzyme preparation. Gel filtration and ion exchange chromatography afforded a pure -amylase (relative molecular mass, 42 000). The pure enzyme was highly active on starch and dextrin. It produced a mixture of oligosaccharides as major products of starch hydrolysis. Maximal activity was reached at 70° C and pH 6.1. Ca²⁺, Na⁺, K⁺ and Sr²⁺ ions stabilized or slightly stimulated the enzyme whereas Ag⁺, Co²⁺, Hg²⁺, Zn²⁺, Cd²⁺ and Fe³⁺ ions strongly inhibited the activity. The enzyme contained 16 amino acids, of which aspartic and glutamic acids were present in the highest proportions. Correspondence to: S. H. Omar     

Classification and characterization of the rice α-amylase multigene family

To establish the size and organization of the rice -amylase multigene family, we have isolated 30 -amylase clones from three independent genomic libraries. Partial characterization of these clones indicates that they fall into 5 hybridization groups containing a total of 10 genes. Two clones belonging to the Group 3 hybridization class have more than one gene per cloned fragment. The nucleotide sequence of one clone from Group 1, OSg2, was determined and compared to other known cereal -amylase sequences revealing that OSg2 is the genomic analog of the rice cDNA clone, pOS103. The rice -amylase genes in Group 1 are analogous to the -Amy1 genes in barley and wheat. OSg2 contains sequence motifs common to most actively transcribed genes in plants. Two consensus sequences, TAACA _G ^A A and TATCCAT, were found in the 5 flanking regions of -amylase genes of rice, barley and wheat. The former sequence may be specific to -amylase gene while the latter sequence may be related to a CATC box found in many plant genes. Another sequence called the pyrimidine box ( _T ^C CTTTT _T ^C ) was found in the -amylase genes as well as other genes regulated by gibberellic acid (GA). Comparisons based on amino acid sequence alignment revealed that the multigene families in rice, barley and wheat shared a common ancestor which contained three introns. Some of the descendants of the progenitor -amylase gene appear to have lost the middle intron while others maintain all three introns.