Production of α-amylase by Myxococcus coralloides D

M.E. FÁREZ-VIDAL, A. FERNANDEZ-VIVAS AND J.M. ARIAS. 1992. Myxococcus coralloides D secreted amylase into a liquid growth medium containing 1% starch. Amylase activity was highest at the end of the exponential growth phase. Of the nitrogen sources tested, the greatest growth and amylase production were obtained with trypticase peptone, casitone, probion L and probion F. When starch was replaced by other carbon sources, amylase production was reduced; trisaccharide produced better results than disaccharide while monosaccharide reduced amylase production to basal levels. Maltose repressed amylase production. Amylase production was greater in stirred flasks, at pH between 6.5 and 7.5, and at temperatures from 28C to 33C. The activity of partially purified M. coralloides D amylase was used to determine the products released from the hydrolysis of starch with thin-layer chromatography, paper chromatography and nuclear magnetic resonance. These products were maltose and glucose and limit dextrins.     

Induction of α-amylase by maltooligosaccharides in Thermomonospora curvata

The action pattern of the α-amylase produced by Thermomonospora curvata is unique. Maltooligosaccharides (maltose to maltopentaose) were tested individually for their ability to induce α-amylase in this thermophilic actinomycete. Maltotetraose was the most inductive followed by maltotriose. Maltose was a good inducer of amylase production when used as sole carbon source, but had relatively little inductive capacity in the presence of either glucose or cellobiose. When cellobiose was added during exponential growth on maltose, maltose utilization and extracellular α-amylase accumulation were transiently inhibited. With maltotriose as the initial carbon source, addition of cellobiose did not inhibit the utilization of the trisaccharide; however, cellobiose, whether added during exponential growth or stationary phase, resulted in the rapid degradation of amylase when maltotriose was depleted from the medium. This inactivation did not appear to be a growth phase-induced phenomenon because stationary phase cells in the absence of cellobiose maintained their peak extracellular amylase level. This cellobiose-mediated α-amylase inactivation would be particularly important during production of the enzyme on a complex lignocellulosic substrate.     

Numerical taxonomy of α-amylase producing Bacillus species

A total of 134 α-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% S_SM). Phenotypic characteristics determined by the API 20E and API 50CHB galleries, other biochemical tests and morphological characteristics were used for the numerical analysis. The API Computer Service identified 45% of the isolates. The amylase yields of 16 α-amylase hyperproducing (AHP) isolates were compared with those of seven amylolytic reference and type strains. The AHP isolates were related to Bacillus subtilis, B. licheniformis and 'B. amyloliquefaciens' .     

Coupling of fermentation and microfiltration for α-amylase production from Bacillus amyloliquefaciens

Abstract: A strain of Bacillus amyloliquefaciens secreting α-amylase was cultivated continuously in a fermentor coupled to a filtration unit. Under the tested operating conditions, the maximum flux was 91 m^-2 h^-1 during the first day and 61 m^-2 h^-1 during the 2nd day. The α-amylase retention was around 30%. Compared to a batch process, continuous cultivation with cell recycling led to lower α-amylase concentrations but to a doubling of volumetric productivities.     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

The nucleotide sequence of an α-amylase gene from an alkalopsychrotrophic Micrococcus sp

An alpha-amylase gene from Micrococcus sp. 207 was cloned into Escherichia coli JM101 using the vector pHSG399. The constructed recombinant plasmid pYK63 contained a 4.8 kb chromosomal DNA fragment derived from strain 207 DNA. The cloned amylase isolated from E. coli JM101 (pYK63) produced mainly maltotetraose from starch, and exhibited temperature and pH activity profiles closely similar to those of the enzyme from the original strain. Nucleotide sequence analysis of the cloned DNA fragment revealed one open reading frame containing the gene which consisted of 3312 bp (1104 amino acids). When compared with several other alpha-amylases, three consensus sequences were identified in the region of the active site. About 300 amino acid residues were present both upstream and downstream of the active site region.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

Contribution of aleurone layer and scutellum to α-amylase synthesis and secretion in wheat and rice grains

Intact aleurone layers from wheat ( Triticum aestivum L. cv Camp Rémy) and rice ( Oryza sativa L. cv Cigalon) grains both contained and secreted more α-amylase (EC 3.2.1.1.) than did the corresponding scutellar tissues. This discrimination was already evident at the earliest stages of germination at which the tissues could be isolated, and became more pronounced upon subsequent germination and growth. Isoenzyme patterns obtained upon isoelectric focusing showed a considerable polymorphism of the α-amylases of each cereal. The enzyme polymorphism pattern was the same in the aleurone layer and in the scutellum, but some secondary constituents appeared to be more specific for the one or the other of the tissues. Moreover, the isozymes found in the tissues were the same as those found to be secreted. A third α-amylase antigen which differs from the well established α₁ and α₁₁ forms was identified in the germinating wheat grains. The presence of Ca²⁺ in the secretion medium favoured maximum secretion of α-amylases from the wheat scutellum and aleurone layers, whereas it inhibited the secretion of the enzymes from the rice aleurone layer.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

Synthesis and localization of α-amylase and α-glucosidase in Bacillus licheniformis grown in batch and continuous culture

In batch and continuous cultures of Bacillus licheniformis NC1B 6346 α-amylase was invariably extracellular and could not be detected in the cytoplasm or cell surface. α-Glucosidase however, was largely intracellular but at the end of exponential growth and during slow growth under Mg²⁺ limitation it was detected in the culture fluid. Both enzymes were susceptible to catabolite repression and glucose totally inhibited their synthesis in batch culture. In maltose-limited chemostat culture, synthesis of both enzymes was maximal at D = 0.2/h and declined at higher growth rates. α-Amylase synthesis was constitutive but α-glucosidase synthesis was induced by maltose and maltotriose but not by methyl-α-D-glucoside or phenyl-α-D-glucoside. α-Amylase was synthesized at pH 6.5 and above in maltose-limited chemostat culture but not below this pH. Intracellular α-glucosidase synthesis varied little with pH. Increasing temperature decreased the synthesis of both enzymes in chemostat culture to the extent that α-glucosidase was undetectable at 50° C. Polar lipid composition varied with pH and temperature but there was no correlation between this and enzyme secretion. Moreover cerulenin, an antibiotic that inhibits protein secretion in some bacteria by interacting with the membrane had no effect on α-amylase secretion but decreased the release of α-glucosidase upon protoplast formation.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Biochemical and immunological characterization of α-amylase isoenzymes of Araucaria araucana

Seven α-amylase isoenzymes present in quiescent seeds of the South American conifer Araucaria araucana were purified by affinity chromatography and partially characterized. The molecular masses of these isoenzymes were 45.7, 47.0, 50.2, 51.2, 52.0, 53.5 and 55.2 kDa. The two main isoforms were separated from each other and from the rest of the isoenzymes by anion-exchange chromatography using a linear gradient of 0 to 0.6 M NaCl and slightly different CaCl₂ concentrations. All isoenzyme bands stained with periodic acid/dansylhydrazine, suggesting that they are glycoproteins. Electroblotting of the isoenzymes onto polyvinylidene difluoride membranes allowed determination of the amino acid composition and NH₂-terminal sequence of the 53.5-, 50.2-and 47.0-kDa isoenzymes. Amino acid compositional analysis demonstrated that these enzymes are rich in glycine, aspartic acid/asparagine, alanine, serine, proline and glutamic acid/glutamine. The NH₂-terminal sequences of the three isoenzymes are identical. Comparison of the amino acid compositions and the NH₂-terminal sequence of these isoenzymes with the cereal and Vigna radiata α-amylases demonstrated that there is no relation between them. However, polyclonal antibodies generated against barley α-amylase cross-reacted with all the A . araucana α-amylases. Peptide mapping analysis of the isoenzymes using cyanogen bromide suggests that there are genetic differences between them.     

Suppression of growth defects of α-amylase secreting Escherichia coli by signal sequence fusion

Abstract In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae . In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.     

Biochemical characterization of α-amylase of the Sunn pest, Eurygaster integriceps

The α‐amylase in the midgut and salivary glands of Eurygaster integriceps was isolated and characterized. The specific activity of α‐amylase in the midgut was 1.77 U/mg protein and in the salivary glands was 1.65 U/mg protein. Sodium dodecylsulfate electrophoresis showed that both midgut and salivary glands contain isozymes. Only a trace amount of α‐amylase activity was detected in the first nymphal stage (0.19 U/mg protein), whereas α‐amylase activity was highest in the third nymphal stage (1.21 U/mg protein). The results show that α‐amylase activity in the immature stages increase constantly to the third instar stage. There was no significant difference in enzyme activity between the third, fourth and fifth nymphal stages and adults. The optimum pH and temperature for the enzyme activity was determined to be 6.5 and 35°C, respectively. The enzyme activity was inhibited by addition of ethylenediaminetetraacetic acid, urea, sodium dodecylsulfate and Mg²⁺, but NaCl and KCl enhanced enzyme activity.     

Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.     

Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.     

Metabolic regulation of rice α-amylase and sucrose synthase genes in planta

Isolated rice embryos were used to investigate the regulatory effects of endosperm extracts and pure sugars on the expression of alpha-amylase gene RAmy3D and a sucrose synthase gene homologous to the maize isozyme Ss2. The high-level expression of RAmy3D in the scutella of isolated embryos could be inhibited by a variety of sugars as well as endosperm extracts from germinated rice grains. Glucose, at a concentration of 250 mM, was most effective in repressing RAmy3D mRNA accumulation. Furthermore, this repression was reversible. Interestingly, RAmy3D repression was always accompanied by the induction of sucrose synthase gene expression. These results support a model in which the expression of alpha-amylase and sucrose synthase genes in the rice scutellum are counter-regulated by the influx of sugars from the endosperm.