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

Production and Characterization of Phospholipases C from some Bacillus thuringiensis Isolates Recovered from Egyptian Soil

Two hundred and thirty one isolates, with the characteristic morphology of Genus Bacillus, were recovered from 100 soil samples collected from 7 different Egyptian governorates, and were screened for phospholipase C (PLC) production by egg-yolk plate method. Sixty isolates have shown very high PLC production and were further assessed using chromogenic assay method. The highest five producers, identified by 16S rRNA gene sequencing as Bacillus thuringiensis, were selected and their PLCs were purified to homogeneity using ammonium sulfate precipitation and Sephadex G-75 gel filtration chromatography. PLCs had molecular masses of 28.5 kDa as indicated by SDS-PAGE. The characteristics of the studied five PLCs were having maximal activities at 35-45°C and pH 7.2. The enzymes could retain more than half of their maximum activities at 30-60°C and pH 7-8. Equivalent activities were recorded at low water tension. PLC from B. thuringiensis KT159186 was relatively thermostable with a maximum activity at 40°C. The half-inactivation temperature was above 50°C, which compared favorably to that of other enzymes. Activity at the wide temperature range (20-80°C) was high (about 50% of maximum),. This PLC could tolerate pH as high as 12 with only 30% loss of activity. Specificity pattern of PLC from the same isolate showed equivalent activities toward phosphatidylcholine and phosphatidylinositol in addition to marked activity toward phosphatidylethanolamine, which makes it a typical non-specific PLC for industrial purposes. In conclusion, these characteristics of PLC from the test isolate make it attractive for various industrial applications

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