Production and characterization of extracellular and intracellular tannase from newly isolated Aspergillus aculeatus DBF 9.

A comparative study on the simultaneous production of extra and intracellular tannase was made from newly isolated fungal strain Aspergillus aculeatus DBF 9. This strain produced five times more intracellular enzyme within 24 h in liquid culture than the extracellular form. Maximum tannase production occurred in the culture broth containing 1-2% (w/v) tannic acid and 0.05-0.1% (w/v) glucose. The pH and temperature optima of both the enzymes were found at 5.0 and 50-60 degrees C, respectively. Extra and intracellular tannase showed good stability at higher temperature, pH values and salt (NaCl) concentration. These properties make the enzyme suitable for pollution control and bioprocess industry.     

Purification and characterization of tannin acyl hydrolase from Aspergillus niger MTCC 2425

The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico-chemical characterization of this enzyme. the extraction of enzyme protein. However, extraction of fungal pigments and proteins was observed to have high pH dependence, and maximum enzyme extraction was obtained at pH 5.5. The two-step purification protocol gave 51-fold purified enzyme with a yield of 20%. The total tannase activity was made up of nearly equal activity of esterase and depsidase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of purified tannase protein indicated it to be made up of two polypeptides of molecular weight 102 and 83 kDa. Based on the Michaelis-Menten constant (Km) of tannase for three substrates tested, tannic acid was the best substrate with Km of 2.8 x 10(-4) M, followed by methyl gallate and propyl gallate. The inhibition was maximum for CaCl2 (58%) whereas EDTA had no modulatory effect on tannase activity. The inhibitor binding constant (KI) of CaCl2 was 5.9 x 10(-4) M Homogenization and detergent pretreatments did not have any remarkable effect on and the inhibition was of noncompetitive type.     

Biodegradation of tannic acid by Citrobacter freundii isolated from a tannery effluent.

A bacterial strain capable of utilizing tannic acid as sole carbon source was isolated from the effluent of a tannery and was identified as Citrobacter freundii. This organism could grow at concentrations as high as 5% (w/v) of tannic acid and produced extracellular tannase to hydrolyze the same. When grown in minimal medium containing 1% tannic acid (w/v) at 30 degrees C, this strain produced 1.87 U/ml of tannase at 6 h. At that time, tannic acid degradation products, namely glucose and gallic acid, were detectable in the culture filtrate; the other intermediate metabolites formed were pyrogallol (extracellular) and pyruvate (intracellular). 2-hydroxymuconic acid is presumed to form as a result of ortho-cleavage of pyrogallol. The proposed biochemical pathway for the degradation of tannic acid by Citrobacter freundii is: Tannic acid-->[Glucose + Gallic acid]-->Pyrogallol -->2-hydroxymuconic acid -->[?]-->Pyruvate.     

Production of an extracellular thermohalophilic lipase from a moderately halophilic bacterium, Salinivibrio sp. strain SA-2

Fifty strains of moderately halophilic bacteria were isolated from various salty environments in Iran. A strain designated as SA-2 was shown to be the best producer of extracellular lipase and was selected for further studies. Biochemical and physiological characterization along with 16S rDNA sequence analysis placed SA-2 in the genus Salinivibrio. The optimum salt, pH, temperature and aeration for enzyme production were 0.1 M KCl, pH 8, 35 degrees C and 150 rpm, respectively. The enzyme production was synchronized bacterial growth and reached a maximum level during the early-stationary phase in the basal medium containing 1 M NaCl. Triacylglycerols enhanced lipase production, while carbohydrates had inhibitory effects on it. The maximum lipase activity was obtained at pH 7.5, 50 degrees C and CaCl(2) concentration of 0.01 M. The enzyme was stable at pH range of 7.5-8 and retained 90% of its activity at 80 degrees C for 30 min. Different concentrations of NaNO(3), Na(2)SO(4), KCl and NaCl had no affect on lipase stability for 3 h. These results suggest that the lipase secreted by Salinivibrio sp. strain SA-2 is industrially important from the perspective of its tolerance to a broad temperature range, its moderate thermoactivity and its high tolerance to a wide range of salt concentrations (0-3 M NaCl).     

Biosynthesis of tannase and gallic acid from tannin rich substrates by Rhizopus oryzae and Aspergillus foetidus

Modified solid-state fermentation (MSSF) of tannin-rich substrates for production of tannase and gallic acid was carried out using two fungal cultures, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557). The tannin rich substrates included powdered fruits of Terminalia chebula and Caesalpinia digyna pod cover powder. The different environmental parameters for the maximum production of tannase and gallic acid were optimized through media engineering. The highest yield of tannase and gallic acid was obtained after 60 h in case of Rhizopus oryzae and after 72 h by Aspergillus foetidus with 3 ml of induced inoculum. The optimum initial pH of the fermentation was found to be 4.5 in case of Rhizopus oryzae and 5.0 for Aspergillus foetidus. MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity. Collectively, the data reveal the potential of the modified solid-state fermentation process for the production of tannase and gallic acid from tannin-rich substrates with R. oryzae and A. foetidus.     

Tannic acid resistance in ruminal streptococcal isolates

Tannin degrading isolates of Streptococcus spp. from rumen of non-adaptive cattle, when grown in BHI broth, were able to tolerate tannic acid upto a level of 50 g/l. An increase in lag period from 1.5 to 6 h was observed for the isolates in presence of increased concentration of tannic acid. In addition, the morphology of gram positive diplococci converted to an elongated chain of 40-50 cells with increasing tannic acid from 1 to 4%. Qualitatively, the tannase activity was found to be present in the isolates tested, indicating their potential of being a tannin degrader.     

Neuraminidase production by a Streptococcus sanguis strain associated with subacute bacterial endocarditis.

The properties of an extracellular neuraminidase produced by a Streptococcus sanguis strain (isolated from a confirmed case of subacute bacterial endocarditis) during growth in a defined medium was examined in this investigation. This enzyme, isolated from concentrated culture supernatants of S. sanguis biotype II, was active against human alpha-1 acid glycoprotein, N-acetylneuramin lactose, bovine submaxillary mucin, and fetuin. Neuraminidase production paralleled bacterial growth in defined medium and was maximal in the early stationary phase of growth but decreased dramatically, probably owing to protease production, during the late stationary phase. The enzyme was purified to near homogeneity by a combination of salt fractionation, ion-exchanged chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 174.4 mumol of sialic acid released per min per mg of protein against human alpha-1 acid glycoprotein. The Km value for this enzyme with human alpha-1 acid glycoprotein as substrate was 2.5 X 10(-3) M, and the enzyme possessed a pH optimum of 6.5. The S. sanguis neuraminidase had a molecular weight of approximately 85,000 as estimated by gel filtration and approximately 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at temperatures of 4 and 37 degrees C for 3 h, but approximately 50% of the enzymatic activity was lost within 30 min at 50 degrees C, with 100% of the enzymatic activity being destroyed within 10 min at temperatures of greater than or equal to 65 degrees C.     

Properties of extracellular neuraminidase produced by group A streptococcus.

Extracellular neuraminidase production by group A streptococci was examined in 92 strains. Fourteen of these strains produced appreciable amounts of enzyme; 12 of the neuraminidase-producing strains belonged to T types 1, 4, and 12. Production of the enzyme paralleled bacterial growth in culture and was maximal in medium containing 0.2% glucose. The enzyme produced by one of these strains was partially purified by ammonium sulfate fractionation and filtration on G-200 Sephadex. Its molecular weight was estimated at 90,000. Activity was optimal at pH 5.7 and in the presence of 0.01 to 0.03 M calcium and magnesium cations. The enzyme was stable at temperatures of 4 and 37 degrees C for at least 24 h but was inactivated within 10 min at temperatures of 50 and 65 degrees C. The enzyme hydrolyzed 40% of the sialic acid in bovine submaxillary mucin, but was inactive on sialyl-lactose, porcine submaxillary mucin, oligosaccharides derived from porcine mucin, or human orosomucoid. The Km value for this enzyme with bovine submaxillary mucin as substrate was in the order of 3.6 x 10(-4) M.     

Some factors affecting endo-β-1,4-glucanase production by two Bacillus strains isolated from Zimbabwean hot springs

Two Bacillus strains capable of producing endo-β-1,4-glucanase enzyme were isolated from Zimbabwean hot springs. Strain HR68 produced the enzyme when cultured using both carboxymethylcellulose (CMC) and simple sugars as carbon source while strain CH43 produced the enzyme on CMC only. The optimum conditions for enzyme production were an initial pH of 6 and a temperature of 50 °C for strain HR68 while for strain CH43 the optimum conditions for enzyme production were an initial pH of 5 and a temperature of 50 °C. A fall in enzyme production was observed after about 4 to 8 h of growth for both isolates. The fall in production coincided with a rise in proteolytic activity in cultures of both isolates. Strain HR68 produced endo-β-1,4-glucanase when grown in continuous culture using glucose as the carbon source.     

Nutritional Requirements for Synthesis of Heat-Labile Enterotoxin by Enterotoxigenic Strains of Escherichia coli

Optimal growth conditions have been established for production of heat-labile enterotoxin (LT) by both porcine and human strains of enterotoxigenic (ENT⁺) Escherichia coli. There were no unusual growth factor requirements, and some strains produced fairly high levels of LT in a basal salts medium containing 0.5% glucose if the pH was carefully controlled. Several amino acids markedly stimulated LT synthesis when added to the basal salts-glucose medium. Methionine and lysine were the most stimulatory for both human and porcine strains. Either aspartic acid or glutamic acid further enhanced LT synthesis in the presence of methionine and lysine, with aspartic acid being more stimulatory for porcine strains and glutamic acid more stimulatory for human strains. There were no apparent vitamin requirements and no unusual cations needed for toxin synthesis except that Fe³⁺ was slightly stimulatory for porcine strains. The stimulation by Fe³⁺ was observed only in the presence of the three amino acids, suggesting that the effect was indirect rather than on toxin synthesis. The carbon source also influenced the yield of LT. Glucose supported maximal synthesis, but other carbon sources which exhibit a high degree of catabolite repression also supported high levels of synthesis. Little or no LT was released below pH 7.0; therefore, because the pH drops during growth from 7.5 to 6.8, even in highly buffered media, it was necessary to adjust the pH to 8.0 to effect complete release of cell-associated toxin. The defined medium containing three amino acids reduced the amount of UV-absorbing material in culture supernatants about fivefold and increased LT activity for various strains from two- to fivefold over a complex Casamino Acids-yeast extract medium. Conditions found to be optimal for synthesis of LT were inhibitory for the heat-stable enterotoxin.     

Thermostable,salt-tolerant α-amylase from Bacillus sp. MD 124

A bacterial strain (MD 124) isolated from municipal garbage and identified as Bacillus sp. was found to be capable of producing salt tolerant and thermostable α-amylase. Rhamnose and peptone were found to be the best carbon and nitrogen source for the production of enzyme. The pH and temperature optima for the enzyme activity were found to be at 6 and 90 °C. Seventy five per cent enzyme activity was retained in 5 M NaCl over 24 hrs.     

Nature of the Kanagawa phenomenon of Vibrio parahaemolyticus.

In a study of the Kanagawa phenomenon of Vibrio parahaemolyticus, both Kanagawa-positive and -negative strains were found to produce hemolytic factors that could not be differentiated on Wagatsuma blood agar. The presence of fermentable carbohydrates in media containing high concentrations of NaCl promoted the growth of V. parahaemolyticus and resulted in a marked decrease in medium pH and increased hemolysin production. The Kanagawa hemolysis of test strains differed according to the carbohydrates added. Clearly defined Kanagawa hemolysis was observed in blood agars of high salt content, but the distinction was lost in media containing 3% NaCl. From the results of this study, the Kanagawa hemolysis was interpreted as an expression of quantitative difference in hemolysin production, a conclusion that is clearly demonstrated on special blood agar of high salt content.     

Illustration of extensive extracellular matrix at the epithelial-mesenchymal interface within the renal stem/progenitor cell niche

Background

Stem/progenitor cells are promising candidates to treat diseased renal parenchyma. However, implanted stem/progenitor cells are exposed to a harmful atmosphere of degenerating parenchyma. To minimize hampering effects after an implantation investigations are in progress to administer these cells within an artificial polyester interstitum supporting survival. Learning from nature the renal stem/progenitor cell niche appears as a valuable model. At this site epithelial stem/progenitor cells within the collecting duct ampulla face mesenchymal stem/progenitor cells. Both cell types do not have close contact but are separated by a wide interstitium.

Methods

To analyze extracellular matrix in this particular interstitium, special contrasting for transmission electron microscopy was performed. Kidneys of neonatal rabbits were fixed in solutions containing glutaraldehyde (GA) or in combination with cupromeronic blue, ruthenium red and tannic acid.

Results

GA revealed a basal lamina at the ampulla and a bright but inconspicuously looking interstitial space. In contrast, GA containing cupromeronic blue exhibits numerous proteoglycan braces lining from the ampulla towards the interstitial space. GA containing ruthenium red or tannic acid demonstrates clouds of extracellular matrix protruding from the basal lamina of the ampulla to the surface of mesenchymal stem/progenitor cells.

Conclusions

The actual data show that the interstitium between epithelial and mesenchymal stem/progenitor cells contains much more and up to date unknown extracellular matrix than earlier observed by classical GA fixation.

     

Biomass production and secretion of hydrolytic enzymes are influenced by the structural complexity of the nitrogen source in Fusarium oxysporum and Aspergillus nidulans.

The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.     

Agar media that indicate acid production from sorbitol by oral microorganisms.

Two varieties of agar medium (Trypticase [BBL Microbiology Systems]-serum-sorbitol-bromcresol purple agar [TSSB] and Trypticase-blood-sorbitol-CaCO3 agar [TBSCa]) indicating microbial acid production from sorbitol were tested. The media were devised for use in studies on the prevalence of sorbitol-fermenting human oral microorganisms incubated in an anaerobic or microaerophilic atmosphere containing 5 to 6% CO2. TSSB contains bromcresol purple as the pH indicator and NaHCO3 as the main buffering salt. TBSCa contains CaCO3 as both the buffering salt and the indicator of acid production. The growth yield of pure cultures of oral microorganisms on TBSCa was shown to equal that on blood agar incubated under similar conditions. TSSB inhibited the growth of several bacteria to various extents. The recovery of sorbitol-fermenting microorganisms from oral specimens was the greatest when the specimens were assayed with TBSCa. The poorer results obtained with TSSB were mainly due to the decoloration of the pH indicator in this medium and the presence of greater numbers of sorbitol false-positive colonies.     

The effects of culture media on the production of xyIan-degrading enzymes by Streptomyces chattanoogensis UAH 23

The production of 4 extracellular xylanolytic enzymes, arabinofuranosidase, endoxylanase, β-xylosidase and acetyl esterase during the growth of Streptomyces chattanoogensis UAH 23 in basal salts media containing different carbon sources was examined. The highest enzyme activities were generally detected when arabinoxylan was used as the carbon source, with the exception of acetyl-esterase where greatest activity was detected when ball-milled straw was used as carbon source. A comparison of enzyme activities revealed that the specific activity of endoxylanase (3.15 U/mg protein) was greater than those of the other three enzyme activities (44.11,5.77 and 2.04 mU/mg protein for acetyl-esterase, a-L-arabinofuranosidase and β-xylosidase, respectively). Cellulase activity could not be detected throughout the study Characterization of the endoxylanase activity revealed that two optima pH could be detected (7.0 and 8.0) and was stable at alkaline pH values between 6.5 and 8.5. The optimum temperature was 55°C with the enzyme stable for one hour at 60°C. Activity staining of non denatured polyacrylamide gels containing culture supernatants revealed the presence of two active endoxylanase bands. The result suggest that two endoxylanase enzymes with different pH optimas are secreted by Streptomyces chattanoogensis UAH 23.     

A cold-active extracellular metalloprotease from Pedobacter cryoconitis--production and properties

An extracellular protease from Pedobacter cryoconitis, isolated from alpine cryoconite on glacier ice, was purified and characterized. Despite high cell densities at a temperature range of 1-25 degrees C, the optimum temperature for protease production was 15 degrees C. Maximum enzyme production was achieved when the strain was grown in a pH-neutral medium containing soybean meal, wheat flour and citrate over 72 h. The 27-kDa enzyme was a metalloprotease (sensitive to EDTA, EGTA and phenanthroline) and showed maximal activity towards azocasein at 40 degrees C and pH 8. The protease was stable for 60 min at 20-30 degrees C, lost 50% of activity after 30 min at 40 degrees C, and was inactivated at 50 degrees C, but was resistant to repeated freezing and thawing. Calcium ions had no protective effect against thermal denaturation. More than 80% of the maximum activity were retained at a pH in the range of 7-10. No activity loss was detected after 1 h at pH 7-9 and 20 degrees C, nor after 1 h of incubation with 3 M urea or 0.1% perborate.     

Nuclear localization of non-specific acid phosphatase(s) activity in rat vaginal epithelium

The nuclear localization of non-specific acid phosphatase(s) in rat vaginal epithelial cells (VEC) by histochemical and biochemical techniques has been reported in the present work. By different histochemical methods, the enzyme activity was predominantly localized in certain nuclei but not in all of them, however, within the nucleus nucleoli are free from the enzyme activity. This enzyme is diffused in the nucleoplasm and perhaps tightly associated with the nuclear proteins. Very little activity was seen in the cytoplasm in the selective population of VEC. After 12 h treatment of estradiol 17beta in vivo the VEC lose the enzyme activity in the basal and intermediate layers, and the activity remains within the nuclei of luminal cells. Enzyme assay in low and high salt extracts of the isolated nuclei of the VEC with or without the enzyme inhibitors indicates that this enzyme may be present in isoforms in the VEC. No activity was observed in these cells at alkaline pH.     

Isolation, characterization and beneficial effects of rice associated plant growth promoting bacteria from Zanzibar soils

This study was undertaken to isolate and characterize plant growth promoting bacteria (PGPB) occurring in four soils of Zanzibar, Tanzania as well as to evaluate their potential use as biofertilizers for rice. A total of 12 PGPB strains were isolated from rice and studied for growth characteristics, carbon/nitrogen source utilization patterns using QTS-24 kits, phosphate solubilization, indole acetic acid (IAA) production, antibiotic resistance patterns and growth at different pH, temperature and salt concentrations. All the isolates were motile and gram negative except Z3-4. Acetylene reduction activity was detected in all isolates ranging from 5.9-76.4 nmole C2H2 reduced/h x mg protein while 9 isolates produced IAA ranged from 20-90.8 mg/l. Most of the isolates showed resistance against different environmental stresses like 10-40 degrees C temperature, 0.2-1 M salt concentration and 4-8.5 pH range. Only one isolate Z2-7 formed clear zones on Pikovskaia's medium showing its ability to solubilize phosphates. Z3-2 was used to develop fluorescent antibodies to check the cross reactivity of the isolates. Inoculation of these bacterial isolates resulted in higher plant biomass, root area, and total N and P contents on Tanzanian rice variety BKN PRAT3036B under controlled conditions. Bacillus sp. Z3-4 and Azospirillum sp. Z3-1 are effective strains and, after further testing under field conditions, can be used for inoculum production of rice in Tanzania. The plant growth promoting effects of these PGPRs suggest that these can be exploited to improve crop productivity of rice in Tanzania.     

Factors influencing β-galactosidase activity of Aeromonas caviae

Aeromonas caviae, often reported to be associated with diarrhoeal patients, elaborates several virulence factors as well as catabolic enzymes such as xylanase and β-galactosidase. Studies on the kinetics of growth of A. caviae and synthesis of β-galactosidase suggested that the activity was cell associated and reached a peak during the late logarithmic phase of growth. The optimum pH for β-galactosidase activity was 7.0 and required Ca²⁺ and glutathione for enhancement of its activity; IPTG also slightly improved the activity. Aerobic cultivation of A. caviae in LB containing glucose, fructose, maltose and sucrose completely inhibited the activity possibly due to acetic acid production. Addition of 100 mM cAMP to the media containing glucose (0.25%, w/v) restored the relative activity by 8.8%; however, the final pH of the media remained acidic. Aerobic growth of A. caviae with other carbon sources did not affect β-galactosidase activity, probably as there was no acid production and thereby the final pH of the media unaltered. Arabinose, xylose and galactose induced the A. caviae β-galactosidase activity by several folds and lactose moderately enhanced its activity.