Optimization of the industrial production of bacterial aamylase in Egypt. V. Analysis of kinetic data for enzyme production by two strains of Bacillus amyloliquefaciens

A kinetic study was conducted for a-amylase production process in shake flasks by the wild type strain of Bacillus amyloliquefaciens (strain 267) and in the fermentor by the amplified variant of the sameorganism (strain 267CH). a-Amylase was produced concurrently with growth up to about 72 h, after which it continued without increase in biomass and, in the case of strain 267 even after biomassdeclined. Application of logistic, Luedeking-Piret and the modified Luedeking-Piret mathematical models to the kinetic data revealed that a-amylase production in both cases occurred through bothgrowth- and non-growth associated mechanisms and that the amount of enzyme produced through non-growth associated mechanism exceeded that produced through growth associated mechanism by 3.5 and 2.3 fold by strains 267 and 267CH, respectively. Although with both strains substrate consumption continued even after growth leveled, the application of the model revealed that the major portion of substrate consumption occurred during growth but that a considerable amount was also consumed after maximum growth was reached, mainly for enzyme production. In the bioreactor, increasing aeration from 1 to 2 vvm increased the overall specific growth rate, the production rate, thespecific production rate, and the specific substrate consumption rate and also shortened the time necessary for maximum production of both biomass and enzyme. The increase in biomass either by prolonging the incubation time or by increasing aeration was accompanied by an increase in enzyme production. However, even after maximum biomass was reached, enzyme production still continued to increase, under both conditions

Optimization of the industrial production of bacterial alpha amylase in Egypt. IV. Fermentor production and characterization of the enzyme of two strains of Bacillus subtilis and Bacillus amyloliquefaciens

Production of alpha amylase using amplified variants of Bacillus subtilis (strain SCH) and of Bacillus amyloliquefaciens (strain 267CH) was conducted in a bioreactor with multiprotein-mineral media. Thetime course of fermentation in a bioreactor revealed that the highest yield (about 8 x 104 U/ml within 60 h) by strain SCH was obtained by applying: 3.5% initial starch, 2% additional starch after 19 h, 3 vvmaeration and 300 rpm agitation. The highest yield (about 19 x 104 U/ml within 100 h) by strain 267CH was obtained by applying: 2.5% initial starch, 2% additional starch after 24 h, 3 vvm aeration, and 300 rpmagitation with the productivity after 60 h reaching only about 14 x 104 U/ml. Production occurred in both the logarithmic and postlogarithmic phases of growth. Maximum consumption of starch and proteinoccurred during the first day of incubation. The optical density peak coincided with enzyme production peak in case of strain SCH and preceded that of enzyme production in case of strain 267CH. The alphaamylase produced by the two strains was shown to be of the liquefying and not the saccharifying type. Both enzymes liquefied starch to a dextrose equivalent of about 15 - 17 at 95oC hence they areclassified among thermostable alpha amylases. They exhibited broad pH and temperature activity profiles. The optimum pH for activity was 4 - 7 for alpha amylase produced by strain SCH and 4 - 8 foralpha amylase produced by strain 267CH while the optimum temperatures for their activities were in the range 37 -75oC at 0.5% starch and in the range 85 - 95oC at 35% starch

Improvement of Bioconversion of Vitamin D3 into Calcitriol by Actinomyces hyovaginalis through Protoplast Fusion and Enzyme Immobilization

Protoplast fusion and enzyme immobilization techniques were applied to increase calcitriol production from vitamin D3 using Actinomyces hyovaginalis, a local isolate recovered from Egyptian soil, that has a potential bioconversion activity of vitamin D3 into calcitriol. A total of sixteen protoplast hybrids, formed between Actinomyces hyovaginalis isolate and two Bacillus species (B. thuringiensis and B. weihenstephanensis) were screened for vitamin D3 bioconversion activity. Compared to wild type isolate, four hybrids (formed between Actinomyces hyovaginalis isolate and B. thuringiensis) were found to preserve the bioconversion activity; out of which, three hybrids coded V2B, V3B and V8A exhibited higher calcitriol production. The hybrids coded V2B and V8A produced, per 1 L culture medium, about 0.5 and 0.4 mg calcitriol corresponding to 350% and 280%, respectively, increase compared to the wild type isolate. Among different alginate concentrations applied, immobilization of cell lysate of Actinomyces hyovaginalis isolate using 2% alginate showed 140% increase in calcitriol production from vitamin D3 compared to the free cell lysate. Activity of the immobilized form was preserved for five repetitive uses over a period of 15 days but with a 50% decline in production occurring at the fifth use

Antimicrobial, Antibiofilm and Immunomodulatory Activities of Lactobacillus rhamnosus and Lactobacillus gasseri against some Bacterial Pathogens

In this study, two Lactobacillus (LAB) strains namely, Lactobacillus rhamnosus EMCC 1105 (L. rhamnosus) and Lactobacillus gasseri EMCC 1930 (L. gasseri) were tested for their antagonistic activities against Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) which are known to be frequently implicated in biofilm infections. The acidic cell free culture supernatant (CFS) of 24 h and 48 h cultures of both LAB stains showed antimicrobial effects against the three pathogens in radial diffusion assay. These effects were abolished upon neutralization of CFS indicating that this effect was due to acids only. Both LAB strains could effectively inhibit the biofilm formation of the three test pathogens and largely replaced them on polystyrene surfaces as demonstrated by crystal violet staining, viable count and scanning electron microscopy. Both of the tested LAB strains could inhibit the protease productivity of S. aureus in 24 h and 48 h dual species-biofilms. The supernatant of 24 h-dual biofilms of P. aeruginosa with L. gasseri also showed a significantly lower protease activity compared to that of P. aeruginosa individual biofilm. Neither LAB strains affected phospholipase C production by the test pathogens when they co-exist during biofilm formation. The different preparations of LAB strains caused no significant change in the levels of gamma interferon expressed by peripheral blood mononuclear cells in response to stimulation by the test pathogens in vitro. In conclusion, L. gasseri and L. rhamnosus can be considered as promising tools for combating biofilm infections

Biophysical Studies of the Membrane-Embedded and Cytoplasmic Forms of the Glucose-Specific Enzyme II of the E. coli Phosphotransferase System (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-IIGlc-YFP and MBP-IIGlc-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET-) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET+). The monomeric species could form a heterodimeric species (FRET+) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-IIGlc activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-IIGlc retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-IIGlc indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-IIGlc

Prevalence of MDR pathogens of bacterial meningitis in Egypt and new synergistic antibiotic combinations.

The aim of this study was identifying bacterial pathogens involved in meningitis, studying their antibiotic resistance profiles, investigating the antibiotic resistance genes as well as evaluating the use of various antibiotic combinations. Antibiotic susceptibility tests were evaluated according to CLSI guidelines. Antibiotic combinations were evaluated by calculating the Fractional Inhibitory Concentration (FIC) index. A total of 71 bacterial isolates were recovered from 68 culture positive CSF specimens. Sixty five of these isolates (91.5%) were recovered from single infection specimens, while 6 isolates (8.4%) were recovered from mixed infection specimens. Out of the 71 recovered isolates, 48 (67.6%) were Gram-positive, and 23 (32.4%) were Gram-negative. Thirty one of the Gram positive isolates were S. pneumoniae (64.6%, n = 48). Out of the recovered 71 isolates; 26 (36.6%) were multidrug-resistant (MDR) isolates of which, 18 (69.2%) were Gram-negative and 8 (30.8%) were Gram-positive. All MDR isolates (100%) showed resistance to penicillin and ampicillin, however, they showed lower resistance to meropenem (50%), levofloxacin (50%), amikacin (48%), pipercillin-tazobactam (45.8%). Most common antibiotic resistance genes were investigated including: tem (21.1%), shv (15.8%), ctx-m (15.8%) coding for TEM-, SHV, CTX-M extended-spectrum beta-lactamases (ESBLs), respectively; aac(6')-I b(26.3%) coding for aminoglycoside 6'-N-acetyltransferase type Ib ciprofloxacin resistant variant; and qnrA (5.3%) gene coding for quinolone resistance. The DNA sequences of the respective resistance genes of some selected isolates were PCR amplified, analyzed and submitted to the GenBank database under the accession numbers, KX214665, KX214664, KX214663, KX214662, respectively. The FIC values for ampicillin/sulbactam plus cefepime showed either additive or synergistic effect against ten tested Gram-negative MDR isolates, while doxycycline plus levofloxacin combination revealed synergism against two MDR Gram-positive isolates. The results indicate high prevalence of antibiotic resistance among MDR isolates. Therefore, new guidelines should be implemented in Egypt to rationalize the use and avoid the misuse and abuse of antimicrobial agents

Experimental and bioinformatics study for production of l-asparaginase from Bacillus licheniformis: a promising enzyme for medical application

Abstract A Bacillus licheniformis isolate with high l-asparaginase productivity was recovered upon screening two hundred soil samples. This isolate produces the two types of bacterial l-asparaginases, the intracellular type I and the extracellular type II. The catalytic activity of type II enzyme was much higher than that of type I and reached about 5.5 IU/ml/h. Bioinformatics analysis revealed that l-asparaginases of Bacillus licheniformis is clustered with those of Bacillus subtilis, Bacillus haloterans, Bacillus mojavensis and Bacillus tequilensis while it exhibits distant relatedness to l-asparaginases of other Bacillus subtilis species as well as to those of Bacillus amyloliquefaciens and Bacillus velezensis species. Upon comparison of Bacillus licheniformis l-asparaginase to those of the two FDA approved l-asparaginases of E. coli (marketed as Elspar) and Erwinia chrysanthemi (marketed as Erwinaze), it observed in a cluster distinct from- and with validly predicted antigenic regions number comparable to those of the two mentioned reference strains. It exhibited maximum activity at 40 °C, pH 8.6, 40 mM asparagine, 10 mM zinc sulphate and could withstand 500 mM NaCl and retain 70% of its activity at 70 °C for 30 min exposure time. Isolate enzyme productivity was improved by gamma irradiation and optimized by RSM experimental design (Box–Behnken central composite design). The optimum conditions for maximum l-asparaginase production by the improved mutant were 39.57 °C, 7.39 pH, 20.74 h, 196.40 rpm, 0.5% glucose, 0.1% ammonium chloride, and 10 mM magnesium sulphate. Taken together, Bacillus licheniformis l-asparaginase can be considered as a promising candidate for clinical application as antileukemic agent

Isolation, screening and improvement of rhamnolipid production by <em>Pseudomonas</em> isolates

611-619Rhamnolipids (RLs) have attracted attention of many scientists because of their low toxicity, biodegradability and ecological acceptability. Nevertheless,their use is extremely limited due to the high cost when compared with chemical surfactants.Therefore,in this studya total of twenty four Pseudomonas isolates were recovered from various soil samples. Screening for RLs production was done using Siegmund Wagner agar plates where six isolates appeared to produce RLs. Isolate P6 was chosen as the most promising RL producer and identified using 16S rRNA sequencing as P. aeruginosa. The best production of 2.5 g/L was obtained by isolate P6 after six days of incubation at 30°C, 250 rpm. Random mutagenesis using UV or gamma radiation resulted in improved RL production by the mutants, the best of which was designated 15GR, which produced RL at concentrations two times higher that of the parent isolate. Rhamnolipids from this mutant showed improved activity and higher emulsifying power relative to that of the parent. Therefore, P6 isolate and its mutant 15GR are promising RL producers which have the advantage of producing RLs from glucose without induction with hydrophobic carbon sources

Rhamnolipid production by a gamma ray-induced Pseudomonas aeruginosa mutant under solid state fermentation

Abstract Solid-state fermentation has a special advantage of preventing the foaming problem that obstructs submerged fermentation processes for rhamnolipid production. In the present work, a 50:50 mixture of sugarcane bagasse and sunflower seed meal was selected as the optimum substrate for rhamnolipid production using a Pseudomonas aeruginosa mutant 15GR and an impregnating solution including 5% v/v glycerol. Using Box–Behnken design, the optimum fermentation conditions were found to be an inoculum size 1% v/v, temperature 30 °C and unlike other studies, pH 8. These optimized conditions yielded a 67% enhancement of rhamnolipid levels reaching 46.85 g rhamnolipids per liter of impregnating solution, after 10 days, which was about 5.5 folds higher than that obtained by submerged liquid fermentation. Although maximum rhamnolipids concentration was obtained after 10 days of incubation, rhamnolipids concentration already reached high levels (41.87 g/l) after only 6 days. This rhamnolipid level was obtained in a shorter time and using lower carbon source concentrations than most studies reported so far. The findings obtained indicate an enormous potential for employing solid-state fermentation for rhamnolipid production by the studied isolate

OXA-48 Carbapenemase-Encoding Transferable Plasmids of Klebsiella pneumoniae Recovered from Egyptian Patients Suffering from Complicated Urinary Tract Infections

Gram-negative bacteria are common causes of urinary tract infections (UTIs). Such pathogens can acquire genes encoding multiple mechanisms of antimicrobial resistance, including carbapenem resistance. The aim of this study was to detect the carbapenemase-producing ability of some Gram-negative bacterial isolates from urine specimens of patients suffering from complicated UTIs at two vital tertiary care hospitals in Cairo, Egypt; to determine the prevalence of carbapenemase genes among plasmid-bearing isolates; and explore the possibility of horizontal gene transfer to other bacterial species. The collected isolates were subjected to antimicrobial susceptibility testing, phenotypic analysis of carbapenemase production, and molecular detection of plasmid-borne carbapenemase genes, then the extracted plasmids were transformed into competent E. coli DH5α. A total of 256 Gram-negative bacterial clinical isolates were collected, 65 (25.4%) isolates showed carbapenem resistance of which 36 (55.4%) were carbapenemase-producers, and of these 31 (47.7%) harbored plasmids. The extracted plasmids were used as templates for PCR amplification of blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP carbapenemase genes. The blaOXA-48 gene was detected in 24 (77.4%) of the tested isolates while blaVIM gene was detected in 8 (25.8%), both blaKPC and blaNDM genes were co-present in 1 (3.2%) isolate. Plasmids carrying the blaOXA-48 gene from 4 K. pneumoniae clinical isolates were successfully transformed into competent E. coli DH5α. The transformants were carbapenemase-producers and acquired resistance to some of the tested antimicrobial agents as compared to untransformed E. coli DH5α. The study concluded that the rate of carbapenem resistance among Gram-negative bacterial uropathogens in Cairo, Egypt is relatively high and can be transferred horizontally to other bacterial host(s)