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.     

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' .     

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.     

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.     

The Formation, General Characteristics and Production of α-Amylase in Heterocaryons and Diploids of Aspergillus oryzae

S ummary . Heterocaryons and diploids from Aspergillus oryzae were investigated with respect to nuclear number/conidium and to conidial size. Heterocaryons usually had larger conidia and more nuclei/conidium than diploids and the haploid parent mutants. Diploids contained significantly fewer nuclei/conidium than haploids. However, they could not be distinguished from haploids by measurement of conidial size. The strains were examined for the production of α-amylase. All auxotrophic mutants produced less α-amylase than the prototrophic wild type. Heterocaryons gave yields which were intermediate between that of their parent mutants or the same as the best producing parent. Diploids which produced more α-amylase than the best producing parent strain were synthesized. The highest yield from a diploid was of the same order of magnitude as the yield from the wild type.     

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.     

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.     

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.     

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, 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.     

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.     

Differential expression of two β-amylase genes of rye during seed development

The expression of two β-amylase loci was analysed in the developing seeds of two inbred lines of rye (Secale cereale L.), one of which was a β-amylase deficient mutant. Enzymatic activity and the contents of enzymatic protein and mRNA specific for each of an endosperm-characteristic and ubiquitous β-amylase were determined throughout the course of caryopsis development. Both loci were expressed in the developing normal line caryopses according to different temporal and quantitative patterns. The ubiquitous enzyme-specific locus β-Amy 2 was expressed earlier; both mRNA and enzymatic protein accumulated to a maximum extent at 10 to 15 days after pollination. In contrast, the highest content of mRNA for endosperm β-amylase (encoded by the β-Amy I locus) was found 20 days after pollination, and the corresponding enzymatic protein accumulated throughout seed development. The expression of the β-Amy I locus was 30- to 40-fold higher than that of the β-Amy 2 locus in terms of maximum specific mRNA accumulation. The expression product of only the β-Amy 2 locus was found in the developing mutant line caryopses. The expression pattern of this locus was similar in the developing normal and mutant line seeds in terms of the temporal accumulation of mRNA and enzymatic protein. However, an approximately 4-fold higher level of ubiquitous β-amylase-specific mRNA was found in the mutant than in the normal line caryopses, and the content of ubiquitous β-amylase protein decreased to near zero at seed maturity in the mutant line, but not in the normal line, caryopses. The enzymatic activities of both β-amylases appeared to be regulated at the level of accumulated enzymatic protein.     

Effects of spermidine on the synthesis of α-amylase in cotyledons of mung bean seedlings

When cotyledons of mung bean [ Vigna radiata (L.) Wilczek] were treated with spermidine (3 m M ) during the first 6 h of imbibition, the development of α-amylase activity in cotyledons during the following 3 days was severely inhibited (75%) This inhibition was due to a slower accumulation of α-amylase protein, which in turn resulted from an inhibition of α-amylase synthesis. The rise in the level of α-amylase mRNA in cotyledons was also inhibited by spermidine treatment. However, the degree of inhibition of mRNA accumulation (40%) was not so marked as that of the activity of α-amylase synthesis (80%). These results are discussed in relation to the mode of action of spermidine on α-amylase expression.     

Nucleotide sequence of the extracellular α-amylase gene in the yeast Schwanniomyces occidentalis ATCC 26077

The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.