Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis

AmyA, an alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal alpha-1,4-glycosidic bonds in various alpha-glucans at 85 degrees C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some alpha-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.     

Temperature impacts the multiple attack action of amylases

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148.

An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.     

Immobilization of mold and bacterial amylases on silica carriers

The immobilization of alpha-amylase and glucoamylase was investigated by several coupling methods on silica carriers, different types of Silokhroms, and silica gels. The most active immobilized mold and bacterial alpha-amylases and mold glucoamylase were obtained with titanium salts. These activities were twice the value of that obtained by glutaraldehyde or azo coupling. The half-lives of A. oryzae alpha-amylase, B. subtilis alpha-amylase, and A. niger glucoamylase, immobilized to silica carriers at 45 degrees C and under continuous operation at a high concentration of substrate, were 14, 35, and 65 days, respectively.     

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.     

Action of glucoamylase from Aspergillus niger on phosphorylated substrate

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger was purified to be free from alpha-amylase and phosphatase (glucose 6-phosphate as substrate). The phosphatase was well separated from the glucoamylase by phosphocellulose ion-exchange chromatography. The glucoamylase action was prevented by the esterified phosphate groups of the substrate. Thus, the extensive action of the glucoamylase on potato starch exposed the 6-posphorylglucosyl residue of the starch at the non-reducing terminal and large molecular weight limit dextrins remained. The concomitant action of the phosphatase was necessary for the complete degradation of the starch.     

Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii

Aspergillus kawachii produces two kinds of alpha-amylase, one is an acid-unstable alpha-amylase and the other is an acid-stable alpha-amylase. Because the quality of the shochu depends strongly on the activities of the alpha-amylases, the culture conditions under which these alpha-amylases are produced were examined. In liquid culture, acid-unstable alpha-amylase was produced abundantly, but, acid-stable alpha-amylase was not produced. The acid-unstable alpha-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no alpha-amylase activity was recognized in the medium. Instead, the alpha-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable alpha-amylase. Usually, acid-unstable alpha-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of alpha-amylase were produced. The production of acid-stable alpha-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.     

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Acid-stable and acid-unstable alpha-amylases of the mold fungi Aspergillus]

Acid-sable alpha-amylase of Asp. niger and acid-unstable, alpha-amylase of Asp. oryzae were studied. It was demonstrated, that beside being a more acid-stable properties, alpha-amylase Asp. niger has increased thermal stability as compared to alpha-amylase Asp. oryzae. The molecular weights of acid-stable alpha-amylase and acid-unstable alpha-amylase are 58 000 and 51 000, respectively. The amino acid composition, and the C- and N-terminal amino acids of both forms of alpha-amylases were determined. It was demonstrated, that the enzymes under study contain one sylfhydryl group per mole of enzyme, which in the Ca2+-bound form plays an important role in the maintenance of the catalytically active enzyme conformation.     

Study of the inhibition of four alpha amylases by acarbose and its 4IV-alpha-maltohexaosyl and 4IV-alpha-maltododecaosyl analogues

Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose. The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different alpha-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic alpha-amylases. The three inhibitors showed mixed, noncompetitive inhibition, for all four alpha-amylases. The acarbose inhibition constants, Ki, for the four alpha-amylases were 270, 13, 1.27, and 0.80 microM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively. The G6-Aca and G12-Aca analogues are the most potent alpha-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known alpha-amylase inhibitors.     

Purification and properties of alpha-amylases in morphological variants of Bacillus subtilis

Extracellular alpha-amylases were isolated from the culture medium filtrates of Bacillus subtilis R-623 morphological variants R, P and S by means of biospecific chromatography on artificial sorbents and then purified to homogeneity. Some properties of purified alpha-amylases were being studied. The molecular weight of alpha-amylases from Bacillus subtilis variants R, P and S equals 57,000, 58,000 and 56,000, and the isoelectric points are at pH 5.4, 5.6 and 5.1, respectively. pH optimum for alpha-amylase from variants R and P is 4.5, and for that from variant S--5.0. alpha-Amylases from Bacillus subtilis R-623 morphological variants are thermostable enzymes. According to the properties studied, they correspond to Bacillus subtilis alpha-amylases that were isolated and described by other researchers.     

Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23

The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Immunochemical studies on alpha-amylase. 3. Immunochemical relationships among amylases from various microorganisms

Sirishinha, Stitaya (University of Rochester School of Medicine and Dentistry, Rochester, N.Y.), and Peter Z. Allen. Immunochemical studies on alpha-amylase. III. Immunochemical relationships among amylases from various microorganisms. J. Bacteriol. 90:1120-1128. 1965.-Immunochemical relationships among amylases obtained from a selected group of microorganisms were examined, and a cross-reaction was detected between the alpha-amylases of Bacillus stearothermophilus and B. subtilis. Immunodiffusion and quantitative precipitin studies, as well as cross-neutralization tests, indicate that B. stearothermophilus alpha-amylase reacts with a portion of antibody present in antisera to crystalline B. subtilis alpha-amylase. Amylases from these two species thus have some aspects of structure in common. Limited data obtained by immunodiffusion suggest that groupings which confer cross-reactivity to the B. stearothermophilus enzyme are lost after exposure to mercaptoethanol in the presence of ethylenediamine-tetraacetate, followed by treatment with iodoacetamide. With the antisera employed and within the concentration range examined, no immunochemical cross-reaction was observed among amylases from Aspergillus oryzae, B. subtilis, B. polymyxa, B. macerans, Pseudomonas saccharophila, and Euglena sanguinis. Immunoelectrophoresis of partially purified B. stearothermophilus alpha-amylase by use of antiserum to the crude enzyme, together with localization of amylase activity in immunoelectrophoretic plates, suggests that B. stearothermophilus alpha-amylase is antigenic in the rabbit.     

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54

AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.     

α-Amylase-catalysed synthesis of alkyl glycosides

Alkyl glycosides were synthesised from starch and alcohols using Aspergillus oryzae α-amylase as catalyst. In the degradation of starch by α-amylase, the alcohols competed with water as glycosyl acceptors. In the reaction with methanol, methyl maltoside and methyl maltotrioside were the main alcoholysis products. Conversion of 45 g/l starch in 30% methanol resulted in a product mixture containing 26 mM maltooligosaccharides and 3.6 mM methyl glycosides. With ethanol, propanol and butanol, alkyl maltosides and alkyl maltotetraosides were detected, and with benzyl alcohol, benzyl glycosides having two, three or five glucose units were formed. No alcoholysis reaction occurred with hexanol or octanol. In conclusion, α-amylase is promising for the one-step synthesis of alkyl glycosides having more than one monosaccharide unit, which are difficult to synthesise in other ways.     

Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases

Molecular mechanisms of irreversible thermal inactivation of two bacterial alpha-amylases, from the mesophile Bacillus amyloliquefaciens and from the thermophile Bacillus stearothermophilus, have been elucidated in the pH range of relevance to enzymatic catalysis. At pH 5.0, 6.5, and 8.0, B. amyloliquefaciens alpha-amylase irreversibly inactivates due to a monomolecular conformational process, formation of incorrect (scrambled) structures which subsequently undergo aggregation. At the last pH, this process can be suppressed by the presence of the substrate starch and consequently a covalent process, deamidation of asparagine and/or glutamine residues, becomes the cause of loss of enzymatic activity at 90 degrees C. Monomolecular conformational scrambling is the predominant cause of irreversible inactivation of B. stearothermophilus alpha-amylase at 90 degrees C at pH 5.0, 6.5, and 8.0. At pH 6.5 another contributing inactivation mechanism is the deamidation of amide residues, and at pH 8.0, O2 oxidation of the enzyme's cysteine residue.     

Purification by expanded bed adsorption and characterization of an alpha-amylases FORILASE NTL from A. niger

In this work the purification and biochemistry characterization of alpha-amylases from Aspergillus niger (FORILASE NTL) were studied. The effects of expansion degree of resin bed on enzyme purification by expanded bed adsorption (EBA) have also been studied. Residence time distributions (RTD) studies were done to achieve the optimal conditions of the amylases recovery on ion-exchange resin, and glucose solution was used as a new tracer. Results showed that height equivalent of the theoretical plates (HETP), axial dispersion and the Prandt number increased with bed height, bed voidage and linear velocity. The adsorption capacity of alpha-amylases, on the resin, increased with bed height and the best condition was at four-expansion degree. alpha-Amylase characterization showed that this enzyme has high affinity with soluble starch, good hydrolysis potential and molecular weight of 116 kDa.