Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone

AIM: Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. METHODS AND RESULTS: The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. CONCLUSIONS: The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. SIGNIFICANCE AND IMPACT OF THE STUDY: A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Characteristics of an extracellular protease isolated from<Emphasis Type="Italic">Bacillus subtilis</Emphasis> AG-1 and its performance in relation to detergent components

An extracellular protease isolated fromBacillus subtilis AG-1 was investigated with respect to various detergents and formulation components. The enzyme had optimum at pH 8.0 and 60 °C temperature while zymographic study revealed two activity bands of 24.9 and 18 kDa. It showed high stability towards non-ionic (Tween 20, Tween 80, Triton X-100) and anionic surfactants sodium dodycyl sulfate (SDS), retaining 100 and 71% of its original activity. Another distinctive feature of the enzyme was its efficient stability towards hydrogen peroxide (H₂O₂) and sodium perborate and different commercial detergent brands. AG-1 protease was also examined for its activity/performance in combination with different stabilizers like glycerol, propylene glycol and polyethylene glycol (PEG). Enzyme showed a promising activity in the presence of this polyols especially PEG (8000). Whilst its compatibility with different commercially available powder and liquid detergents was also very interesting. These results suggest AG-1 protease as a good detergent compatible and can be utilized in the formulation of an environment friendly bio-detergent.     

Geobacillus thermoleovorans subsp. stromboliensis subsp. nov., isolated from the geothermal volcanic environment

A novel thermophilic, aerobic, endospore-forming bacterium, designated strain PizzoT, was isolated from geothermal volcanic environment. Samples were collected from the Pizzo sopra la Fossa site at Stromboli Island (Eolian Islands, south of Italy) at the high altitude of 918 m. Cells of strain PizzoT were rod-shaped and stained Gram-positive. Growth was observed between 50 and 75 degrees C (optimum 70 degrees C) and at pH 5.0-8.0 (optimum pH 7.0). NaCl (0.4%, w/v) supported growth and among the hydrocarbons tested none induced growth. The G+C content of the DNA was 54.1 mol% and the sequence analysis of the 16S rRNA gene showed that the new isolate was phylogenetically closely related to the members of the Bacillus rRNA Group 5. DNA-DNA hybridization studies revealed a borderline similarity between the new isolate and Geobacillus thermoleovorans DSM 5366T (69.8%) and Geobacillus kaustophilus DSM 7263T (63.4%). On the basis of phylogenetic analysis and physiological traits of the isolate, it should be described as a new member of the Geobacillus thermoleovorans species and it is proposed that strain PizzoT can be classified as Geobacillus thermoleovorans subsp. stromboliensis, subsp. nov. (ATCC BAA-979T; DSM 15393T).     

Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria

An extracellular protease from a newly isolated seawater haloalkaliphilic bacterium, haloalkaliphilic bacteria Ve₂-20-9₁ [HM047794], was purified and characterized. The enzyme is a monomer with a 37.2 kDa estimated molecular weight. It catalyzed reactions in the pH range 8–11 and performed optimally at pH 10. While maximal activity occurred at 50 °C, the temperature profile shifted from 50 to 80 °C in 1–3 M NaCl. The enzyme's thermal stability was probed using circular dichroism (CD) spectroscopy with NaCl at 50 and 70 °C. The changes in the enzyme's secondary structure were also analyzed using Fourier transform infrared spectroscopy (FTIR). The N-terminal amino acid sequence GKDGPPGLCGFFGCI exhibited low homology with other bacterial proteases, which highlights the enzyme's novelty. The enzyme was labile in anionic surfactant (1% w/v SDS) but showed stability in non-ionic surfactants (Tween 20, Tween 80 and Triton X-100 all 1% v/v), commercial detergents, and oxidizing and reducing agents. The enzyme's excellent stability in commercial detergents highlights its potential as a detergent additive.     

Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations

The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0–11.0 and 65–70 °C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 °C.

The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 °C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 °C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

Improving production of hyperthermostable and high maltose-forming alpha-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications

By cultivating Geobacillus thermoleovorans in shake flasks containing cane molasses medium at 70 degrees C, the fermentation variables were optimized by 'one variable at a time' approach followed by response surface methodology (RSM). The statistical model was obtained by central composite design (CCD) using three variables (cane-molasses, urea and inoculum density). An overall 1.6- and 2.1-fold increase in enzyme production was achieved in the optimized medium in shake flasks and fermenter, respectively. The alpha-amylase titre increased significantly in cane-molasses medium (60 U ml(-1)) as compared to that in the synthetic medium (26 U ml(-1)). Thus the cost of enzyme produced in cane molasses medium (0.823 euros per million U) was much lower than that produced in the synthetic starch-yeast extract-tryptone medium (18.52 euros per million U). The shelf life of bread was improved by supplementing dough with alpha-amylase, and thus, the enzyme was found to be useful in preventing the staling of bread. Reducing sugars liberated from 20% and 30% raw pearl millet starch were fermented to ethanol; ethanol production levels attained were 35.40 and 28.0 g l(-1), respectively.     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.     

On the properties of agar gel containing ionic and non-ionic surfactants

Rheological and thermal properties of agar sol and gel in presence of various cationic, anionic and non-ionic surfactants are reported. The agar used was from the red seaweed Gelidiella acerosa. The gel strength, viscosity, rigidity (G'), gelling temperature and melting temperature were observed to decrease in presence of non-ionic surfactants whereas these were enhanced in presence of ionic surfactants. TGA studies showed that 1.5% agar gels containing non-ionic surfactants lose water at a lower temperature than the control agar gel whereas gels containing ionic surfactants hold on to water more tenaciously. DSC studies, on the other hand, show that the gel to sol transition occurs at lower temperatures in presence of non-ionic surfactants and at higher temperature in presence of ionic surfactants when compared with the control gel. The non-ionic surfactants, Triton X-100 and Brij 35, enabled relatively concentrated agar extractive to be filtered readily, as a result of which water usage in the process could be reduced by 50%. The surfactant was subsequently removed through freeze-thaw operations to restore the gelling capacity of the agar. The finding that 0.3-0.4% (w/v) sodium lauryl sulfate (SLS) lowers the sol-gel transition temperature from 41 to 36 degrees C without adversely affecting gel strength is another useful outcome of the study that may enable better formulations of bacteriological agar to be prepared.     

Alkali-thermostable and cellulase-free xylanase production by an extreme thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>

The optimization of cultural variables resulted in a marked enhancement in the secretion of cellulase-free and alkali-thermostable xylanase (EC 3.2.1.8) by an extreme thermophile Geobacillus thermoleovorans. The enzyme secretion was enhanced when the medium was supplemented with xylan (0.15%) and Tween-80 (0.1% v/v). In wheat bran-tryptone medium, the peak in enzyme production was attained within 42 h in a fermenter as compared to 72 h in shake flasks. Optimization of the culture conditions resulted in a 7.72-fold enhancement in enzyme production. The cellulase-free xylanase was optimally active at pH 8.5 and 80°C, and it was found to be useful in the pre-bleaching process of paper pulps.     

Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp.]

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.     

Short communication: Bacillus cereus capable of degrading SDS shows growth with a variety of detergents

A strain of Bacillus cereus was isolated from a detergent-polluted pond. This strain showed growth with exceedingly high concentration of both anionic and non-ionic detergents. Detergent such as SDS was rapidly taken up by the cells and degraded to dodecan-1-ol by the enzyme alkylsulphatase.     

Effect of various surfactants (cationic,anionic and non-ionic) on the growth of <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> (NRRL 2999) in relation to aflatoxin production

The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p < 0.05). At a concentration of 0.001%, CDAB was found to enhance toxin production, while SDS was found to inhibit it when compared with other surfactants. Non-ionic surfactants, polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene lauryl ether (Brij-35) and ethoxylated p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.     

Analysis of supercooling activities of surfactants

Supercooling-promoting activities (SCAs) of 25 kinds of surfactants including non-ionic, anionic, cationic and amphoteric types were examined in solutions (buffered Milli-Q water, BMQW) containing the ice nucleation bacterium (INB) Erwinia ananas, silver iodide (AgI) or BMQW alone, which unintentionally contained unidentified ice nucleators, by a droplet freezing assay. Most of the surfactants exhibited SCA in solutions containing AgI but not in solutions containing the INB E. ananas or BMQW alone. SCAs of many surfactants in solutions containing AgI were very high compared with those of previously reported supercooling-promoting substances. Cationic surfactants, hexadecyltrimethylammonium bromide (C16TAB) and hexadecyltrimethylammonium chloride (C16TAC), at concentrations of 0.01% (w/v) exhibited SCA of 11.8 °C, which is the highest SCA so far reported. These surfactants also showed high SCAs at very low concentrations in solutions containing AgI. C16TAB exhibited SCA of 5.7 °C at a concentration of 0.0005% (w/v).     

Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80

A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30 degrees C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 degrees C for 6 h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 degrees C and the half-life of the enzyme at 50 degrees C increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system.     

Novel characteristics of a 33KDa protein (pp33) rapidly phosphorylated in IL3 dependent cells by stimulation with IL3

We report the novel properties of a 33 KDa cellular protein rapidly phosphorylated by stimulation of growth by IL3 in IL3 dependent lines. Although pp33 is readily soluble in SDS, SDS-solubilised pp33 is insoluble in non-ionic detergents and is excluded from electrophoretic analysis (IEF, NEPHGE) employing such detergents. Native pp33 is not extracted by non-ionic detergents with or without cation chelation. pp33 is concentrated in a cell fraction containing endoplasmic reticulum where it is associated with a specific trypsin-sensitive degredative enzyme, active at 4 degrees. Its unusual characteristics and kinetics of phosphorylation suggest pp33 may be a novel molecule, explain its absence in studies elsewhere where non-ionic detergent extraction has been exclusively used and suggest it is intimately related to the signal transduced by IL3.     

α-淀粉酶AmyP对有机溶剂和表面活性剂的耐受性

AmyP是一个来自海洋宏基因组文库的α-淀粉酶。AmyP不仅对log Pow值从4．5到-0．24的各种有机溶剂均具有良好的耐受性,而且能被正辛醇、正辛烷和甲苯提高活性为139％、118％和119％。正辛醇影响AmyP的淀粉水解产物、葡萄糖的含量增加、麦芽三糖的含量降低。非离子型的表面活性剂Tween-20、Tween-80和Triton X-100存在条件下,AmyP的活性反而有不同程度的提高。但是,AmyP对阴离子型的SDS和阳离子型CTAB的耐受性稍差。结果表明AmyP是一个同时具有有机溶剂和表面活性剂耐受性的新型α-淀粉酶。     

Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99

An alkalophilic Aspergillus nidulans KK-99 produced an alkaline, thermostable xylanase (40 IU/ml) in a basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15% N) pH 10.0 and 37 degrees C. The partially purified xylanase was optimally active at pH 8.0 and 55 degrees C. The xylanase was stable in a broad pH range of 4.0-9.5 for 1 h at 55 degrees C, retaining more than 80% of its activity. The enzyme exhibited greater binding affinity for xylan from hardwood than from softwood. The xylanase activity was stimulated (+25%) by Na+ and Fe2+ and was strongly inhibited (maximum by 70%) by Tween-20, 40, 60, SDS, acetic anhydride, phenylmethane sulphonyl fluoride, Triton-X-100. The xylanase dose of 1.0 IU/g dry weight pulp gave optimum bleach boosting of Kraft pulp at pH 8.0 and temperature 55 degrees C for 3 h reaction time.