Numerical taxonomy of alpha-amylase producing Bacillus species

A total of 134 alpha-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% SSM). 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 alpha-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'.     

Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates

Summary Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values of temperature, particle size, moisture, and incubation time within the indicated ranges.     

Comparison of growth rates of two clones ofBacillus subtilis,producer ofα-amylase

In a population of the productiveBacillus subtilis strain the production of α-amylase differentiates at the cell level. Individual cells in the population were analyzed and the production of isolates was tested. The mean specific activity of α-amylase in the productive group after the isolation was 5.0 U/mg dry substance and in the nonproductive group 1.5 U/mg dry substance. This ratio remained unchanged during long-term observations and after repeated transfers. In selected strains of both groups the specific growth rate was determined in a synthetic medium containing various amounts of casein hydrolyzate. The detected differences in the growth rate between the productive and the nonproductive clones are determined by amino acid concentration.     

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.     

Cell growth and α-amylase production characteristics ofBacillus subtilis

Growth, differential rate of α-amylase synthesis and production characteristics ofBacillus subtilis DP 1 (isolate from starch materials) in comparison with 10Bacillus strains were examined in batch fermentation. The effect of the carbon and nitrogen source was evaluated with regard to cell growth and enzyme production. The pH optimum of enzyme activity was 6.5 and temperature optimum 60°C.     

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.     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

Stability of the recombinant plasmid carrying theBacillus amyloliquefaciens α-amylase gene inB. subtilis

Summary Theα-amylase gene ofBacillus amyloliquefaciens has previously been cloned into pUB110 to give the recombinant plasmid, pKTH10 (Palva 1982. Gene 19:81–87). Strains transformed by this plasmid are promising candidates for industrialα-amylase production. The stability of pKTH10 was determined in variousB. subtilis strains possessing specific alleles which affect the level ofα-amylase secretion.B. subtilis strains carrying pKTH10 were cultivated in starch-containing medium for up to 50 generations without antibiotic selection and then screened for the presence of pKTH10. The plasmid proved stable enough (< 1.0% cured after 50 generations) for industrial batchwise enzyme production in two strains, but in asacU9 strain (thesacU9 mutation increases concominantly the production ofα-amylase levansucrase and proteases) 99.9% of cells had lost pKTH10 after 50 generations, although the parental plasmid (pUB110) was stable in this strain (0.09% cured after 50 generations). The instability of pKTH10 in thesacU9 strain seems somehow to be related to high expression of the clonedα-amylase gene: when grown in a medium restrictingα-amylase production, only 0.53% ofsacU9 cells had lost pKTH10 after 50 generations.     

Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis

Abstract The gene coding for the thermostable α-amylase Bacillus licheniformis has been isolated from a direct shotgun in Escherichia coli using the bacteriophage lambda as a vector. The fragment containing the α-amylase gene has been sub-cloned in pBR322 and its restriction map determined. The α-amylase produced by the E. coli clones retained the thermostability of the B. licheniformis enzyme. Expression and properties of the gene product in E. coli and Bacillus subtilis have been examined.     

Protein secretion in Bacillus subtilis as influenced by the combination of signal sequence and the following mature portion

Abstract We have constructed secretion vector plasmids that have the signal sequence of the Bacillus licheniformis penicillinase gene ( penP ) or the Bacillus stearothermophilus α-amylase gene ( amyT ). We have also constructed penP, amyT and hsa (human salivary α-amylase gene) cartridges. Each of these cartridges was cloned on secretion vectors in Bacillus subtilis , and enzyme production was examined. When amyT vector was used, nearly the same efficiency of enzyme secretion was observed for amyT and penP cartridges. When penP vector was used, enzyme secretion for amyT decreased to about 3% of that for penP cartridges. The eukaryotic gene hsa was hardly expressed in any secretion vectors in B. subtilis .     

A polyphasic taxonomic study of thermophilic bacilli from shallow, marine vents

Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics. A numerical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus reference strains by 89 selected features. Results from cluster analysis showed the formation of nine clusters. Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to Geobacillus thermodenitrificans. The remaining isolates grouped together with different reference strains. Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S rDNA sequences of 9 strains. Subsequent DNA/DNA reassociation studies and determination of the base composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G. thermoleovorans and one isolate as Bacillus pallidus. Four isolates represented two novel species of Bacillus. The remaining four represented novel Geobacillus species, one of which has recently been described as Bacillus vulcani DSMZ 13174 T.     

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.     

Characterization of a Thermostable α-Amylase from Bacillus licheniformis NCIB 6346

With one exception (NCIB 9668), the extracellular amylases from 10 strains of Bacillus licheniformis were thermostable and retained more than 98% of their original activity after incubation at 85°C for 60 min. The enzyme from B. licheniformis NCIB 6346 was purified 30-fold by ion-exchange chromatography and was characterized. It had an endo-action on starch yielding maltopentaose as the major product, and was identified as an α-amylase. The purified enzyme had a molecular weight of 62 650, was stable between pH 7 and 10 and was maximally active at 70-90°C at pH 7.0. It closely resembled commercial thermostable α-amylases in its general properties and it is concluded that B. licheniformis provides a good source of these enzymes.     

The carbohydrases of Bacillus stearothermophilus NCIB 11412 and NCIB 10814

Summary Two strains (NCIB 11412 and NCIB 10814) of the thermophilic organism Bacillus stearothermophilus were found to produce complex carbohydrase systems. The enzyme activities in each system include -amylase as the major component, maltase, pullulanase, a minor amylase and cyclodextrinase. The latter three activities are produced in low yield in both strains. A crude enzyme preparation from each strain possessed maltogenic properties on hydrolysis of soluble starch. Following rigorous purification procedures, the purified major -amylase from either strain did not produce maltose as a major end-product of starch hydrolysis. However, a partially purified mixture of pullulanase, minor amylase and cyclodextrinase activities from NCIB 11412 and NCIB 10814 produced 56.4% and 62.0% maltose, respectively, from soluble starch.     

Tyrosine 39 of GH13 <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Thermococcus hydrothermalis</Emphasis> contributes to its thermostability

The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.     

Parametric optimization of extracellular α-amylase production by thermophilicBacillus coagulans

The culture parameters required for optimum production of extracellular α-amylase by the thermophilicBacillus coagulans are described. The optimum pH, temperature and incubation period for amylase production were 7, 50°C and 48 h, respectively. Age of inoculum (48 h) and its level, (2%) were critical for maximum amylase yield. The enzyme secretion was high in rice starch and beef extract as compared to other carbon and nitrogen sources tested. The addition of mustard oil cake (1%) and agitation at 1.7 Hz resulted in an enhancement of α-amylase secretion.     

The occurrence and properties of Bacillus thuringiensis isolated from free-living animals

AIMS: To assess the prevalence and properties of Bacillus thuringiensis isolated from the intestines of small mammals. METHODS AND RESULTS: Bacillus thuringiensis was found in 11% of rodents and 17% of insectivores. Using PFGE of chromosomal DNA, SDS-PAGE of whole-cell proteins and biochemical tests (API system), 12 isolates and three reference strains were classified. Numerical analysis revealed 61% and 89% similarity of protein profiles and biochemical properties of the bacilli, respectively. The results of PFGE were consistent with the outcomes of the analysis of protein profiles. CONCLUSIONS: Although B. thuringiensis is not common in the intestines of small mammals, it is heterogeneous at the genotypic and phenotypic level. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here help to explain the diversity and the ecological significance of B. thuringiensis. Future study should focus on the toxic activity of the isolated strains.     

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.     

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.     

Characterization of Aeromonas hydrophila strains and their evaluation for biodegradation of night soil

Among 67 psychrotrophic bacterial isolates of Leh, India screened for production of hydrolytic enzymes at 10 °C, four belonging to Aeromonas hydrophila were characterized and evaluated for biodegradation of night soil. All strains produced metalloproteases on a variety of carbon and nitrogen sources. Strains LA1 and LA15 also produced α-amylase and PC5 both α- & β-amylase. No amylase was produced by PN7, however it produced lipase. Casein and glucose induced maximum enzyme activity (protease and amylase) in LA15 and PC5, respectively. In LA1, maximum induction of protease was observed with casein and of amylase with maltose. Corn oil/tributyrin served as the best inducers for protease and lipase production by PN7. A. hydrophila strains were found to be psychrotrophic with optimum growth and enzyme activity at 20 and 37 °C, respectively. Maximum biodegradation of night soil was observed by strain LA1 at 5–20 °C. This revised version was published online in July 2006 with corrections to the Cover Date.