Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Functional significance of amylase polymorphism in Drosophila melanogaster. III. Ontogeny of amylase and some α-glucosidases

Changes in amylase (E.C. 3.2.1.1), maltase (E.C. 3.2.1.20), sucrase, and PNPGase activities in relation to changes in wet weight and protein content were studied during the development of larvae and adult flies from two strains of Drosophila melanogaster, homozygous for different amylase alleles. All -glucosidase activities increase exponentially during a large part of larval development, parallel to the increase in weight, and drop at the end of the third instar. Amylase activity of the Amy ¹ strain follows the same pattern. In contrast, amylase activity of the Amy ^4,6 strain continues its exponential increase longer. In the third larval instar amylase activity in the Amy ^4,6 strain becomes much higher than in the Amy ¹ strain. During the first hours of adult life amylase activity of the two strains does not differ. Then Amy ^4,6 activity starts to rise and becomes much higher (4–5 times) than Amy ¹ amylase activity, which remains approximately constant. All adult enzyme activities are much higher than in larvae. Comparison of enzyme activity of amylase and -glucosidases in larvae and adults confirms that differences in amylase activities can become important only when starch is a limiting factor in the food.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express α-amylase and glucoamylase separately or as bifunctional fusion proteins

Eight constructions involving the Bacillus subtilis -amylase gene (amyE), a mouse pancreatic -amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one -amylase and the glucoamylase, two double-cassette plasmids (expressing one or other -amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis -amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic -amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis -amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only -amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes. The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth. The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch.     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Evolution of the response patterns to dietary carbohydrates and the developmental differentiation of gene expression of α-amylase in Drosophila

Intraspecific variation of -amylase activity in D. melanogaster and D. immigrans, which is distantly related to D. melanogaster, and interspecific variation of -amylase activity in 18 Drosophila species were examined. The amount of intraspecific variation of -amylase activities measured in terms of coefficient of variation in D. melanogaster and D. immigrans was one-half and one-tenth or less, respectively, of the interspecific variation in 18 Drosophila species. We also surveyed the response patterns of -amylase activity to dietary carbohydrates at the larval and adult stages. The levels of -amylase activity depended on both repression by dietary glucose (glucose repression) and induction by dietary starch (starch induction). In general, our data suggest that glucose repression was conserved among species at both stages while starch induction was mainly observed in larvae, although the degree of the response depended on species. In D. lebanonensis lebanonensis and D. serrata, larvae expressed electrophoretically different -amylase variants (isozymes) from those of adult flies. These results may suggest that the regulatory systems responsible both for the response to environment and developmental expression are different among species in Drosophila. Correspondence to: T. Yamazaki     

Production of native-starch-degrading enzymes by a Bacillus firmus/lentus strain

Summary A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native starch, resulted in complete degradation of native starch granules from potato, maize and wheat at a temperature of 37°C. Glucose was found as a major product. Production of maltose, maltotriose and maltotetraose was also observed. Native-starch-degrading activity (NSDA) could be selectively adsorbed on potato-starch granules, whereas soluble-starch-degrading activity (SSDA) remained mainly in solution. The use of such a starch-adsorbed enzyme preparation on native starch resulted in a completely changed product pattern. An increase in oligosaccharides concomitant with less glucose formation was observed. An increased conversion of soluble starch to maltopentaose was possible with this starch-adsorbed enzyme preparation. It is concluded that NSDA comes from -amylase(s) and SSDA from glucoamylase(s) and/or -glucosidase(s). Cultivation of B. firmus/lentus on glucose, maltose, or soluble starch resulted in substantially smaller quantities of (native) starch-degrading activity.Offprint requests to: D. J. Wijbenga     

Effect of polyethylene glycol on Bacillus subtilis α-amylase purification with raw and modified starch

Polyethylene glycol was found to enhance adsorption of Bacillus subtilis -amylase on starch in optimum concentration 10 % (w/w). Degree of adsorption at 12°C was increased from 83 to 98 % and from 30 to 81 % for cross-linked and raw starch, resp. Higher sorption capacity and easy desorption of -amylase without temperature or pH change was reached at 22 °C. Yield of -amylase 95 % and purification factor 8.3 were achieved on the cross-linked starch column. The method is suitable for -amylase isolation from PEG phase after its microbial production in aqueous two-phase systems.     

Bacterial diversity in thermophilic aerobic sewage sludge

Summary Using -amylase as an example, extremely thermophilic Bacilli isolated from heat-treated sewage sludge are shown to be a source for enzymes stable and active at high temperatures. The isolates which are classified as subspecies of Bacillus stearothermophilus differ from each other in protein composition indicating the heterogeneiety of that subspecies. Media are evaluated for good growth and high enzyme productivity. Best media are those composed of three or four different complex components like combinations of peptone, soy grist, and malt extract, -amylase production on simple carbon sources is negligible. From the cultivation supernatants crude -amylase extracts are prepared and their behaviour at high temperatures is described. The optimal temperature of all tested enzymes is 80°C. They are stable at suboptimal temperatures for over 20 h and at 95° C 50% of their activity is lost within 2 h. The activity at 95° C is however preserved for over 3 h in presence of starch. The products of the starch digestion are maltotriose, maltose, and some glucose. The amylases can therefore compete in activity and stability with commercially available -amylases from Bacillus licheniformis.     

Molecular cloning of alpha-amylases from cotton boll weevil,Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of -amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two -amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5 and 3 RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7 kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several -amylase inhibitors from plants were assayed against A. grandis -amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and -AI1 inhibitors on A. grandis -amylase activity. This work suggests that genetic engineering of cotton to express -amylase inhibitors may offer a novel route to A. grandis resistance.     

Primer extension studies on α-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression

Relative levels of different -amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI -amylase groups allowed the levels of different -amylase mRNAs to be assessed both within and between the two groups.In all aleurone systems the same set of -amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of -amylase mRNAs. In particular, the regulation of -amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.     

Interaction of hydrolytic and phosphorolytic enzymes of starch metabolism in Kalanchoë daigremontiana

The degradation of starch by a protein fraction of Kalanchoë daigremontiana Hamet et Perrier, obtained by ammoniumsulfate precipitation (30–70%), was found to be catalyzed by -and -amylase (EC 3.2.1.1 and EC 3.2.1.2, respectively) and by starch phosphorylase (EC 2.4.1.1). The activity of these enzymes was determined by chromatographic analysis of the reaction products; separation and identification of -amylase was accomplished by heat-inactivation of -amylase and -glucosidase. When the interaction of amylolytic and phosphorolytic enzymes was comparatively studied, it was found that without inorganic phosphorus in the reaction mixture, ¹⁴C-starch was converted predominantly to maltose and glucose; supplementation with 1–10 mM orthophosphate (Pi) resulted in an increase in glucose-1-phosphate formation and a concomitant reduction of maltose production. Since the total volume of starch degradation remained approximately constant, Pi apparently inhibits -amylase (Ki about 3 mM Pi). Thus, free Pi in the cell participates in the regulation of starch catabolism, serving as a substrate for starch phosphorylase while simultaneously reducing the production of maltose. With respect to glucan synthesis, adenosinediphosphoglucose--1,4-glucosyltransferase (EC 2.4.1.22), maltose phosphorylase and maltoseglucosyltransferase were also found to be active. The last-named enzyme catalyzes an exchange between dextrins and is considered to provide primer carbohydrates for the synthesis of polyglucans.Abbreviations ADPG adenosinediphosphoglucose - G1P glucose-1-phosphate - PEG polyethylenglycol - PEP phosphoenolpyruvate - Pi orthophosphate     

Characterization of Schwanniomyces castellii mutants with increased productivity of amylases

Summary The production of -amylase activity in the yeast Schwanniomyces castellii strain 1402 is repressed in the presence of the non-metabolizable glucose analogue, 2-deoxy-glucose. Selection for resistance to 2-deoxy-glucose after treatment with ethyl methane sulphonate (EMS) or UV light has yielded mutants displaing increased -amylase activities. One such mutant, S. castellii strain 1436, was found to exhibit constitutive -amylase activity in glucose-containing medium. This constitutive enzyme activity was also observed under pilot scale fermentation conditions when the pH was maintained constant at 5.5±0.1. The disaccharide maltose served as a stronger inducer of -amylase activity than the natural substrate starch in both the wild type (1402) and mutant (1436) strains.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Starch degradation by the mould Trichoderma viride I. The mechanism of starch degradation

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of -amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well.Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained.Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable -glucosidase appears to be produced.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Expression of the aglB and aglA Genes in E. coli and the Properties of the Recombinant Enzymes

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85°. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with -glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent -amylase and -glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl₂, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the -glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent -glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with -links. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrare. For the enzyme to be active, the presence of NAD⁺, DTT, and Mn²⁺ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

Effect of gassing,agitation, substrate supplementation and dialysis on the growth of an extremely thermophilic archaeon Pyrococcus woesei

Summary Growth of an extremely thermophilic archaeon, Pyrococcus woesei, at 90°C in a 2–1 fermentor was significantly enhanced by gassing with N₂/CO₂ (95%/5%). Both growth and -amylase activity were also positively influenced by increasing the agitation speed up to 1200 rpm under continuous gassing at 0.2 vvm. However, increasing the agitation speed to 2400 rpm led to decreases in the maximum cell concentration and -amylase activity. Fed-batch cultivation resulted in increases in the specific growth rate, maximum cell concentration and -amylase activity. Although the latter two parameters were higher when the broth was supplemented with both starch and concentrated medium, the specific growth rate was relatively smaller. Cultivation in a dialysis reactor gave a cell concentration of 2 × 10⁹ cells/ml, which represents a 2.8-fold increase over that obtained in ordinary batch cultivation. This increase in the cell concentration was accompanied by a 5.2-fold increase in -amylase activity. Correspondence to: G. Antranikian     

Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium 2021: Effect of carbon sources

Summary Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3 % concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose. glucose and higher dextrins.     

Pullulanase is a characteristic of manyKlebsiella species and functions in the degradation of starch

Summary The type strainsKlebsiella pneumoniae NCTC 9633,K.ozaenae NCTC 5050 andK.rhinoscleromatis NCTC 5046, representative for all members of the genusKlebsiella, were found to produce pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41). In addition, 58 fresh isolates ofKlebsiella sp. of human origin were screened for growth on a defined solid medium with either maltose, maltodextrin mixture, soluble starch, glycogen, or pullulan as the sole carbon source. All of the strains showed luxurious growth on maltose and maltodextrins, seven strains grew poorly or not at all on the polymeric substrates, soluble starch, pullulan or glycogen. Three fresh isolates out of the 51 strains which did grow on each carbon source tested were examined in more detail with respect to a possible involvement of pullulanase in the utilization of -glucans. The production of pullulanase was inducible by growth of the cells on -glucans, whereas cultivation on glycerol, D-glucose or lactose did not lead to enzyme formation. The level of pullulanase activity in the three strains varied under otherwise comparable culture conditions, as did the level of a co-inducible -amylase. Comparative growth experiments on linear or branched -glucans allow the conclusion that the cooperation of hydrolases specific for 1,4--glucosidic linkages (-amylase) and for 1,6--linkages (pullulanase) is an obligatory requirement for the effective utilization of starch and glycogen.