Production and Characteristics of Raw-Potato-Starch-Digesting α-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting α-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30°C. The raw-potato-starch-digesting α-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying α-amylase.     

Comparison of the α-Amylase of Bacillus subtilis and Bacillus amyloliquefaciens

The alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) of Bacillus subtilis strain W23 is less negatively-charged than the alpha-amylase of B. amyloliquefaciens strain F, as determined by electrophoretic mobility in polyacrylamide gel at pH 8.6. The alpha-amylase of strain W23 is immunologically unrelated to the alpha-amylase of strain F, as judged by lack of cross-reaction in Ouchterlony immunodiffusion studies. The pH range of maximal activity for the enzyme of strain W23 was 5.7 to 6.7, with a maximum at 6.3. The pH range of activity for the alpha-amylase of strain F was 5.5 to 6.5, with a maximum at 5.9. No significant difference was found in the effect of temperature on the activity of the alpha-amylase of strain W23 and strain F. alpha-Amylase production by strain W23 occurs throughout the 7-hr growth period, whereas enzyme production by strain F does not begin until the culture enters the stationary phase of growth. The total amounts of enzyme produced by strains W23 and F after 7 hr of growth were 0.3 and 25.5 units/ml, respectively.     

Genetic and Biochemical Studies on Cell-Bound α-Amylase in Bacillus subtilis Marburg

A small but significant amount of alpha-amylase activity was detected in the cells of Bacillus subtilis Marburg. The cell-associated activity was almost constant regardless of the level of extracellular alpha-amylase activity. The cell-bound amylase activity could be separated into three components, upon Sephadex G-75 chromatography, referred to as components A, B, and C. Component C showed the same properties as the extracellular alpha-amylases so far examined. Component A had a molecular weight greater than 70,000, as judged from the elution position on Sephadex G-75, and became smaller upon treatment with trypsin but was still larger than that of component C. An alpha-amylase mutant that lacked extracellular alpha-amylase completely because of a mutation within the structural gene of the enzyme was found to lose all three cell-bound amylase components simultaneously. These data suggest strongly that the cell-bound amylase components are precursors of the extracellular alpha-amylase and that the alpha-amylase of this organism is produced under the direction of the same gene whether the enzyme is within or outside the cell.     

Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg²⁺ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.     

Genetic Control of the Rate of α-Amylase Synthesis in Bacillus subtilis

The level of extracellular alpha-amylase (EC 3.2.1.1) of Bacillus subtilis Marburg was increased about fivefold by introducing the amyR marker from B. natto 1212 through transformation. amyR2 of B. natto 1212 has been assumed to determine a high level of alpha-amylase of the organism. The gene acts specifically on alpha-amylase synthesis but not on the production of other extracellular enzymes. alpha-Amylase of an amyR2-carrying strain was found to be quite similar to that of an isogenic amyR1-carrying strain in the thermostability and electrophoretic behavior of whichever amylase the strain produces. Marburg-type alpha-amylase (amyEm) or B. natto-alpha-amylase (amyEn). Anti-amylase serum titration indicates that a high level of the enzyme activity in the amyR2-carrying strain is caused by the existence of more enzyme rather than the presence of an enzyme having higher efficiency. This is supported further by the fact that amyR controls the synthesis of the amyE gene product in mutant M9, which synthesizes a temperature-sensitive-alpha-amylase, and in mutant M07, which secretes cross-reacting material. The results indicate that amyR regulates the rate of alpha-amylase synthesis.     

Isolation of Mutants Defective in α-Amylase from Bacillus subtilis: Genetic Analyses

The rate of alpha-amylase (EC 3.2.1.1) synthesis in Bacillus subtilis is regulated by a gene, amyR, located near a structural gene, amyE, for the enzyme. To construct a fine map of the amyR-amyE region, we isolated 28 mutants defective in alpha-amylase activity. Eleven mutants out of 28 showed no alpha-amylase activity, whereas the other 17 showed less alpha-amylase activity than the parent. Out of 17 partially positive alpha-amylase mutants, 10 produced temperature-sensitive enzymes, and 4 produced immunologically altered enzymes, two of which are concurrently temperature-sensitive, and 5 produced smaller amounts of alpha-amylases which are indistinguishable from normal enzyme in their temperature sensitivity and immunological properties. Two out of 11 alpha-amylase-negative mutants produced material that cross-reacted with anti-amylase serum, and 3 mutants carried suppressible mutations by the suppressor described by Okubo. Mapping data indicate that all 28 mutation sites are located in the amyE region, and none of the groups of the mutants mentioned above contains lesions that are clustered in a single region of amyE. The amyR gene seems most likely to adjoin the terminal region of amyE.     

Transformation of Bacillus subtilis in α-Amylase Productivity by Deoxyribonucleic Acid from B. subtilis var. amylosacchariticus

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-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.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Introduction of Raw Starch-Binding Domains into Bacillus subtilis α-Amylase by Fusion with the Starch-Binding Domain of Bacillus Cyclomaltodextrin Glucanotransferase

We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 α-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.     

Production of Thermostable α-Amylase, Pullulanase, and α-Glucosidase in Continuous Culture by a New Clostridium Isolate

The production of α-amylase, pullulanase, and α-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60°C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of α-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.     

High Production of Thermostable β-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis

By cloning the β-galactosidase gene of Bacillus stearothermophilus IAM11001 (ATCC 8005) into Bacillus subtilis, enzyme production was enhanced 50 times. β-Galactosidase could be purified to 80% homogeneity by incubating the cell extract of B. subtilis at 70°C for 15 min, followed by centrifugation to remove the denatured proteins. Because of its heat stability and ease of production, β-galactosidase is suitable for application in industrial processes.     

Purification and Characterization of α-Amylase from Bacillus licheniformis CUMC305

α-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90°C and pH 9.0, and 91% of this activity remained at 100°C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60°C, 3 h at 70°C, and 90 min at 80°C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the α-amylase enzyme was fully stable after a 4-h incubation at 100°C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 × 10⁵ J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V_max values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na⁺, Ca²⁺, and Mg²⁺, showed stimulatory effect, whereas Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Ag⁺, Fe²⁺, Co²⁺, Cd²⁺, Al³⁺, and Mn²⁺ were inhibitory. Of the anions, azide, F⁻, SO₃²⁻, SO₄³⁻, S₂O₃²⁻, MoO₄²⁻, and Wo₄²⁻ showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, β-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. α-Amylase was fairly resistant to EDTA treatment at 30°C, but heating at 90°C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu²⁺ and Fe²⁺ but not by the addition of Ca²⁺ or any other divalent ions.     

Intracellular α-Amylase of Streptococcus mutans

Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.     

Properties of Thermosensitive Extracellular α-Amylases of Bacillus subtilis

Enzymological properties of four thermosensitive alpha-amylases (M3, M9, M18, and M20) brought by different mutation sites in alpha-amylase structural gene of Bacillus subtilis were compared with those of the parental alpha-amylase NA64. Two thermosensitive alpha-amylases (M9 and M20) were altered not only in their thermosensitivity but also in their immunological properties, catalytic properties, molecular weights determined by the gel filtration on a Bio-Gel P-100 column, and others. The other two thermosensitive alpha-amylases (M3 and M18) were altered only in their thermosensitivity.     

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.     

Occurrence of an Affinity Site apart from the Active Site on the Raw-Starch-Digesting but Non-Raw-Starch-Adsorbable Bacillus subtilis 65 α-Amylase

α-Cyclodextrin specifically inhibited raw starch digestion by Bacillus subtilis 65 α-amylase. The raw starch digestibility and α-cyclodextrin-Sepharose 6B adsorbability of this α-amylase were simultaneously lost when the specific domain corresponding to the affinity site essential for raw starch digestion was deleted by proteolysis. Occurrence of the affinity site on raw-starch-digesting enzymes was proven also with bacterial amylase.     

Zeamatin Inhibits Trypsin and α-Amylase Activities

Zeamatin is a 22-kDa protein isolated from Zea mays that has antifungal activity against human and plant pathogens. Unlike other pathogenesis-related group 5 proteins, zeamatin inhibits insect α-amylase and mammalian trypsin activities. It is of clinical significance that zeamatin did not inhibit human α-amylase activity and inhibited mammalian trypsin activity only at high molar concentrations.