Effective solubilization and single-step purification of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> α-amylase from insoluble aggregates

A high level expression of thermostable α-amylase gene from Bacillus licheniformis in Escherichia coli was obtained. The recombinant enzyme was mainly produced in the form of insoluble aggregates. The enzyme was solubilized without using denaturing agents and purified to homogeneity in a single step by ion exchange chromatography. The enzyme was purified 138-fold with a final yield of 349 %; the specific activity of the purified enzyme was 1343 U/mg.     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

Affinity chromatography of α-amylase from Bacillus licheniformis

An affinity chromatographic method with a novel eluant from Bacillus licheniformis is described. α-amylase was bound to starch, starch-celite, starch-Sepharose columns and the bound α-amylase was rapidly eluted with 2% (w/v) white dextrin. The binding capacity of α-amylase to starch column is 380 μmol/g of starch. The purified enzyme showed a single polypeptide on SDS-polyacrylamide gel electrophoresis with a molecular weight of 58 kD. The specificity of purified enzyme was confirmed by immunodiffusion, immunoelectrophoresis. Single radial immunodiffusion and western blotting studies analyzed the synthesis of enzyme at different time points.     

Rapid method for the affinity purification of thermostable α-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Summary A rapid and efficient method the exploiting affinity of α-amylase for its substrate starch is described. α-amylase from Bacillus licheniformis was purified to homogeneity by ammonium sulphate precipitation and affinity chromatography with 230-fold purification. The α-amylase adsorption to various starches was examined in order to screen its ability for highest binding to starch. The α-amylase was bound to starch more tenaciously, hence various eluants like maltose, soluble starch and high salts could not elute the bound α-amylase. However, the bound α-amylase was instantly eluted using 2% (w/v) dextrin. The purified enzyme showed a single polypeptide on SDS-PAGE, with a molecular weight of 58 kD. Western blot analysis confirmed the specificity of antibody raised against purified α-amylase.     

Cloning and Expression of Thermostable α-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable α-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more α-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the α-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the α-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80°C for 60 min.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Cloning and expression of the gene encoding <Emphasis Type="BoldItalic">Streptomyces coelicolor</Emphasis> A3(2) α-galactosidase belonging to family 36

The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.     

Thermostable α-amylase from derepressed Bacillus licheniformis produced in high yields from glucose

High yields of thermostable α-amylase was produced by Bacillus licheniformis 44MB82-G, resistant to glucose catabolite repression, on the basis of inexpensive raw materials and glucose as a main carbon source. The optimal parameters for the α-amylase production were an agitation rate of 500 rpm, constant air-flow rate (1 vvm) and cultivation temperature 40°C. An enzyme activity of 4800–5000 U/ml culture medium was reached in 96–120 h. The α-amylase preparation had the following characteristics: α-amylase activity 55 000 U/ml, high thermostability (98% residual α-amylase activity after 10 min treatment at 90°C), protein content 88 mg/ml and dry substances 30%.     

Properties of the recombinant α-glucosidase from Sulfolobus solfataricus in relation to starch processing

An α-glucosidase activity (EC 3.2.1.20) isolated from Sulfolobus solfataricus strain MT-4 was characterised and found of interest at industrial level in the saccharification step of hydrolysis process of starch. The gene encoding for the enzyme was expressed in Escherichia coli BL21 (DE3) with a yield of 87.5 U/g of wet biomass. The recombinant cytosolic enzyme was purified to homogeneity with a rapid purification procedure employing only steps of selective and progressive thermal precipitations with a final yield of 75.4% and a purification of 14.5-fold. The properties of this thermophilic α-glucosidase were compared with those of the α-glucosidase of a commercial preparation from Aspergillus niger used in the starch processing.     

Extracellular calcium-inhibited alpha-amylase of Bacillus coagulans B 49

Extracellular α-amylase (EC 3.2.1.1) from Bacillus coagulans B 49 was purified to homogeneity by ion-exchange chromatography and gel filtration. The optimum pH and temperature for dextrinizing activity were 6–7 and 70°C and for saccharolytic activity were 7 and 60°C, respectively. Calcium inhibited α-amylase activity even at low concentrations (10 m ), and most of its activity could be restored by dialysis against EDTA. Other cations such as Mg²⁺, Fe²⁺, and Hg²⁺ also inhibited amylase activity, while Mn²⁺ exhibited a slight stimulatory effect. The activity of the enzyme was not affected by ethylenediaminetetraacetic acid (EDTA).     

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

The most abundant α-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This α-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an α-amylase by polarimetry, substrate specificity, and end product analyses. The purified α-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This α-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. β-glucan) are not substrates of the enzyme. A very low amount of this α-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or α-amylase which is integrally associated with the chloroplast.     

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds

α-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10⁻⁶ m gibberellic acid (GA₃) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10⁻² m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified α-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of α-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10⁻⁴ m 5-FU prior to the addition of GA₃ resulted in decreased thermal stability of GA₃-induced α-amylase and who interpreted this as evidence that the mRNA for α-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified α-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of α-amylase mRNA synthesis.     

A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.     

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an α-Amylase and a Cyclodextrin-Hydrolyzing Enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.     

Purification, biochemical characterisation and partial primary structure of a new α-amylase inhibitor from Secale cereale (rye)

Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae

Resistance to penicillin in non-β-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42–581) into an ATG expression vector. When the recombinant PBP 2 molecules were over-expressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2′ (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2′ for [³H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other β-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

Expression and Characterization of Glycosylated and Catalytically Active Recombinant Human α-Galactosidase A Produced in Pichia pastoris

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme α-galactosidase A. This enzyme is responsible for the hydrolysis of terminal α-galactoside linkages in various glycolipids. An improved method of production of recombinant α-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human α-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human α-galactosidase A. Recombinant human α-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25°C, but not at the standard growth temperature of 30°C. The recombinant α-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant α-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.     

Adsorption-elution purification of chimeric <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> leucine aminopeptidase II with raw-starch-binding activity

Summary A chimericBacillus stearothermophilus leucine aminopeptidase II (LAPsbd) has been constructed by introducing the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase into the enzyme. LAPsbd was adsorbed onto raw starch and the adsorbed enzyme could be eluted from the adsorbent by soluble starch in 20 mM Tris–HCl buffer (pH 8.0). The adsorption of LAPsbd onto raw starch was affected by raw starch concentration, pH, and temperature, while the temperature and incubation time had no obvious effects on the elution of adsorbed enzyme. The molecular weight of purified enzyme was estimated to be 61 kDa. About 84% of LAPsbd in the cell free extract was recovered through one adsorption–elution cycle with a purification of 20-fold. The high quantity and purity of the recovered enzyme coupled with the easy performance make the adsorption–elution procedure suitable for industrial applications.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation

The biosynthetic mechanism of α-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of α-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.

In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of α-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of α-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of α-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.

The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of α-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of α-amylase were found to consist of three isozymes. The in vitro translated precursor forms of α-amylase consisted of four multiple components. These results indicate that heterogeneity of α-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice α-amylase isozymes are encoded by multiple genes.