β-1, 3-Glucanase Produced by Alkalophilic Bacteria Bacillus No. K-12-5

Bacillus No. K–12–5 isolated from soil produced a β-1,3-glucanase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media, and no growth was observed in neutral media such as nutrient broth. The β-1,3-glucanase of Bacillus No. K–12–5 was purified by DEAE-cellulose, Sephadex G–100 and hydroxyl apatite columns. The enzyme was most active at pH 5.5 ~ 8.0 which was much broader and higher than those of Bacillus criculans enzyme. The sedimentation constant was about 3.6 and molecular weight was about 40,000. The isoelectric point was about pH 3.5 and the enzyme was most stable at pH 7. Calcium ion was not effective to stabilize the enzyme. The enzyme did not hydrolyse laminaritriose. Laminaritetraose was hydrolysed, and glucose and laminaritriose were detected in the hydrolysate. The enzyme split laminaran at random and yielded glucose, laminaribiose, laminaritriose and higher oligosaccharides. If the enzyme is a single entity, it is a type of endo-β-1,3-glucanase. However, activity of hydrolysis of fungal cell walls was lower than that of B. circulans enzyme.     

Xylanase Produced by Alkalophilic Bacillus No. C-59-2

Bacillus No. C–59–2 isolated from soil produced a xylanase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media, and no growth was observed in neutral media such as nutrient broth. The xylanase of this bacteria was purified by CM-celluIose, hydroxyl apatite and Sephadex G–75 columns. The enzyme was most active at pH 5.5~9 which was much broader and higher than those of other xylanases. The sedimentation constant was about 3.5 S and isoelectric point was pH 6.3. The enzyme was most stable at pH 7 and calcium ion was effective to stabilize the enzyme. The enzyme activity was inhibited by Hg²⁺, Ag²⁺ and Cd^{2 +} Maximum hydrolysis rate of xylan by the enzyme was about 40%. The enzyme split xylan and yielded xylobiose and higher oligosaccharides. Therefore, this enzyme is considered to be a type of endo-xylanase.     

Alkaline Catalase Produced by Bacillus No. Ku-1

Bacillus No. Ku-1 isolated from soil produced and alkaline catalase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media. The alkaline catalase in the culture fluid was purified by DEAE-cellulose and Sephadex columns. The enzyme was most active at pH 10.0 and was stable at pH 7.0 to 8.5. The sedimentation constant was about 12.5 S. The enzyme was strongly inhibited by NaN₃, KCN, FeSO₄ and Fe₂ (SO₄)₃. Properties of the enzyme are almost same as those of catalases so far reported except optimum pH for enzyme action and Kat.f. value (4.4×10⁴).     

Isolation and Some Properties of Alkalophilic Bacteria Utilizing Rayon Waste

Bacillus No. C-11 which utilized rayon waste was isolated. This strain belongs to the genus Bacillus from its morphological and biochemical characteristics but grew better in alkaline media than in neutral media. Residual sugars of rayon waste were 34.7 % after 2 days cultivation, 25.5% after 4 days and 7.0% after 9 days. Yeast extract and N-source, such as polypeptone or urea stimulated the utilization of rayon waste. The long period cultivation optimum pH was about 11, and the short period cultivation optimum pH was about 9. Partially purified hemicellulase from Bacillus No. C-11 was most active at pH 7, but still active at pH 10. The stable pH for this enzyme action was in the range of 5.5 to 9, and from the hemicellulose enzymatic digest, xylose, xylobiose, xylotriose and oligosaccharides were detected.     

Thermostable,salt-tolerant amylase fromBacillus sp. 64

A thermostable, salt-tolerant amylase was produced byBacillus sp. 64, with maximum amylase production (8.0 U/ml culture filtrate) after 24-h growth. Partially purified amylase was stable at 60°C for 30 min and 80% of the original activity was retained when incubated in 5m NaCl over 24 h. Starch or dextrin was the best carbon source and peptone the best nitrogen source for the production of the enzyme. Amylase was secreted over a wide pH range (5 to 11) with the maximum activity between pH 7 and 8. Ca²⁺ and Mg²⁺ stimulated growth and enzyme production.NCL Communication No. 5209.     

Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101

Summary An alkalophilic Bacillus sp. no. AH-101, which produced extremely thermostable alkaline protease, was isolated among 200 soil samples. The enzyme production reached its maximum level of 1500 units/ml after about 24 h in alkaline medium (pH 9.5). The enzyme was most active toward casein at pH 12–13 and stable to 10 min incubation at 60° C from pH 5–13. Calcium ions were effective in stabilizing the enzyme especially at higher temperatures. The optimum and stable temperatures were about 80° C and below about 70° C respectively in the presence of 5 mM calcium ions. The enzyme was completely inactivated by phenylmethane sulphonyl fluoride, but little affected by ethylenediaminetetraacetic acid, urea, sodium dodecylbenzenesulphonate and sodium dodecyl sulphate. The molecular weight and sedimentation constant were approximately 30 000 and 3.0S respectively, and the isoelectric point was at pH 9.2. These results indicte that no. AH-101 alkaline protease is more stable against both temperature and highly alkaline conditions than any other protease so far reported.     

Identification and Growth Characteristics of α-Galactosidase-producing Microorganisms

Two kinds of α-galactosidase-producing microorganisms, strain No. 31–2 and strain No. 7–5, have been isolated from soil and subjected to a determinative study. On the basis of the morphological and physiological characters, the strain No. 31–2 was identified to be belonged to genus Micrococcus and the strain No. 7–5 to genus Bacillus. The former strain, Micrococcus sp. No. 31–2, produced exclusively an intracellular α-galactosidase, and the latter one, Bacillus sp. No. 7–5, secreted the enzyme into culture medium. The cell growth and enzyme production of both strains were observed to reach the maximum under an alkaline culture condition. The intracellular α-galactosidase of Micrococcus sp. No. 31–2 was inducible by galactose, melibiose, and raffinose, while the α-galactosidase of Bacillus sp. No. 7–5 was produced constitutively.     

Purification and Properties of d-Xylose Isomerase from Alkalophilic Bacillus No. KX-6

An alkalophilic Bacillus No. KX-6 isolated from soil produced a d-xylose isomerase in alkaline media. The striking characteristic of this bacterium was its especially good growth in alkaline media. The d-xylose isomerase of this bacterium was purified by ammonium sulfate fractionation, DEAE-Sepharose ion exchange column chromatography and G-200 gel Alteration. The molecular weight and sedimentation constant were approximately 120,000 and 9.35 S, respectively. The enzyme was most active at pH 7~10 and was stable at pH 6.0 to 11.0. Enzyme activity was stimulated by cobalt ion but inhibited by Hg^{2 +}, Ag^{2 +}, and Cu^{2 +}. Substrate specificity studies showed that this enzyme was active on d-xylose, d-glucose, d-ribose, and d-arabinose. The smaller Km value and larger V_max value for d-xylose indicated that this enzyme is essentially d-xylose isomerase.     

Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Isolation of Denitrifying Photosynthetic Bacteria

An alkaline proteinase produced by Bacillus sp. was purified and crystallyzed through isopropanol or ammonium sulfate precipitation, decolorization with DEAE-cellulose and gel filtration with Sephadex G-100. The optimum pH for caseinhydrolysis was 11.5 and the activity was completely inactivated after incubation with DFP. The specific activity on Hammersten casein was about 4,500 units/mg enzyme protein. The enzyme also hydrolyzed synthetic ester substrate such as Ac-Tyr-OEt, Ac-Phe-OEt or Bz-Met-OMe. The isoelectric point was pH 10.7, and the molecular weight was estimated to be about 17,500 by the sedimentation equilibrium method and 16,000 by gel filtration method. Some physicochemical properties and the amino acid composition of the enzyme were investigated, indicating that the enzyme is distinguishable from alkaline proteinase of Bacillus species so far reported.     

Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06

Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had K_m and V_max values of 3.45 mg mL⁻¹ and 387.3 µmol min⁻¹mg⁻¹, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Modification of α‐Amylase by Sodium Alginate

Sodium alginate, activated by periodate oxidation, was covalently linked to porcine pancreatic α‐amylase via reductive alkylation with NaBH₄. The enzyme‐polymer conjugate, purified by gel filtration on Fractogel EMD BioSEC (S), retained about 50% of the native specific amylolytic activity. The sugar content was estimated to be 712 mol of monosaccharides per mol of enzyme protein. An average of 11 amino groups out of 21 groups from α‐amylase were modified with the polysaccharide. The functional stability was improved for α‐amylase after conjugation with sodium alginate. In comparison with the native enzyme, the thermostability of α‐amylase was increased by this modification. In addition, the stability in the range of pH 5.0–11.0 was improved for the modified enzyme. The conjugate was also more resistant to denaturation by 0.3% sodium dodecylsulphate, retaining about 10% of its initial activity after 120 min of incubation. The formation of stabilizing salt bridges in the protein surface of the α‐amylase‐polysaccharide complex was confirmed by FT‐IR spectrometry. Attending to the results obtained, we conclude that the covalent attachment of the anionic polysac‐charide sodium alginate to the enzymes might be a useful and non‐expensive method for improving the stabilization of these biocatalysts under various denaturing conditions.     

Properties of an extracellular amylase purified from a Bacillus species

A strain of Bacillus produced an amylase with properties characteristically different from known bacterial amylases. The purified 80 kDa protein of pI 5.1 dextrinized starch, glycogen and pullulan. The temperature and pH optima of the enzyme were 60 °C and 6.6 respectively. In the presence of 0.05 M CaCl₂, the enzyme retained stability for 15 min at 80 °C. Antibodies raised to the amylase protein showed no reaction with -amylases of Bacillus sp. and B. licheniformis. In culture, proteolytic degradation of the enzyme was observed.     

Reisolation and growth conditions of Bacillus agar-exedens

Several agarolytic Bacillus strains have been isolated. Their properties agree with those described by Wieringa (1941) for Bacillus agar-exedens. These strains are the first reisolates since the original cultures were lost. A second group of isolates is related to the agarolytic B. palustris var. gelaticus of Sickles and Shaw (1934). B. agar-exedens requires carbohydrates for growth. In mineral-glucose media growth is inhibited by peptone at pH values of about 7 or less. Under alkaline conditions no inhibition by peptone is observed. A method for the enrichment of B. agar-exedens is described.     

Effect of amino compounds on alkaline amylase production by alkalophilic Bacillus sp.

Of the amino compounds investigated, β-alanine, dl-norvaline and d-methionine were effective for the production of alkaline amylase by alkalophilic Bacillus no. A-40-2. The addition of 0.5% dl-norvaline and 0.5% d-methionine to the culture medium increased amylase production 1.7-fold, while they repressed growth slightly or strongly. There was no relation between amylase yield and the extracellular protein amount. Because alkaline protease activity was negligible in the culture fluid, these compounds might change the regulation of amylase synthesis and/or make amylase excretion easier.     

Effect of tannins on growth and amylase production by Calvatia gigantea

The effect of tannins was investigated on growth and α-amylase (α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) production by the edible fungal species Calvatia gigantea, grown in a laboratory-scale fermenter on acorn starch media containing up to 2 g tannins l⁻¹. No inhibition of both growth and amylase excretion was observed when the fungus was cultivated on media containing 40 to 100 times higher tannin concentration than that reported to inhibit microbial growth. Amylase excretion was enhanced when starch was dry sterilized but specific growth rate was higher when starch was wet sterilized. Biomass and amylase production increased with increasing substrate concentration and specific growth rate reached its maximum value at 20 g l⁻¹ starch concentration. The optimum pH of biomass and amylase productionwas 5.0–5.5 and 6.0−6.5 respectively and that of temperature was 29–32 and 29–30°C respectively. Maximum yields of 68 250 U amylase and 0.58–0.60 g biomass g⁻¹ acorn were obtained at optimum growth conditions. A plot of reciprocal growth rate vs. reciprocal starch concentration made it possible to calculate K_s = 0.84 g acorn starch l⁻¹ and μ_max = 0.249 h⁻¹.     

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates

Abstract

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10?U/mL, whereas total protein production levels were at 15?mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03?mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

Cloning and Sequencing of an Original Gene Encoding a Maltogenic Amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. US149 Strain and Characterization of the Recombinant Activity

A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases. In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum activity was obtained at 40°C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from β-cyclodextrin, and panose from pullulan.