Production and Secretion of a Recombinant Vibrio parahaemolyticus Chitinase by Escherichia coli and Its Purification from the Culture Medium

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.     

The chitin catabolic cascade in the marine bacterium Vibrio cholerae: characterization of a unique chitin oligosaccharide deacetylase

Chitin, one of the most abundant organic substances in nature, is consumed by marine bacteria, such as Vibrio cholerae, via a multitude of tightly regulated genes (Li and Roseman 2004, Proc Natl Acad Sci USA. 101:627-631). One such gene, cod, is reported here. It encodes a chitin oligosaccharide deacetylase (COD), when cells are induced by chitobiose, (GlcNH(2))(2), or crude crab shells. COD was molecularly cloned (COD-6His), overproduced, and purified to apparent homogeneity. COD is secreted at all stages of growth by induced V. cholerae. The gene sequence predicts a 26 N-terminal amino acid signal peptide not found in the isolated protein. COD is very active with chitin oligosaccharides, is virtually inactive with GlcNAc, and slightly active with colloidal ([(3)H]-N-acetyl)-chitin. The oligosaccharides are converted almost quantitatively to products lacking one acetyl group. The latter were characterized by mass spectrometry (ESI-MS), and treatment with nitrous acid. COD catalyzes the following reactions (n = 2-6): (GlcNAc)(n)--> GlcNAc-GlcNH(2)-(GlcNAc)(n-2) + Ac(-). That is, COD hydrolyzes the N-acetyl groups attached to the penultimate GlcNAc residue. The gene bank sequence data show that cod is highly conserved in Vibrios and Photobacteria. One such gene encodes a deacetylase isolated from V. alginolytics (Ohishi et al. 1997, Biosci Biotech Biochem. 61:1113-1117; Ohishi et al. 2000, J Biosci Bioeng. 90:561-563), that is specific for (GlcNAc)(2), but inactive with higher oligosaccharides. The COD enzymatic products, GlcNAc-GlcNH(2)-(GlcNAc)(n), closely resemble those obtained by hydrolysis of the chitooligosaccharides with Nod B: GlcNH(2)-(GlcNAc)(3-4). The latter are key intermediates in the biosynthesis of Nod factors, critically important in communications between the symbiotic nitrogen fixing bacteria and plants. Conceivably, the COD products play equally important roles in cellular communications that remain to be defined.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization

The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 degrees C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.     

Iron control of the Vibrio fischeri luminescence system in Escherichia coli

Iron influences liminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB ⁺ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICD ABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di (o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB ⁺ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of -galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8–9 fold each for tonB, 2–3 fold each for tonB ⁺) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.     

Physiological characteristics of recombinant Escherichia coli cells displaying poly-His peptides

Using the Escherichia coli OmpC protein as an anchoring motif, four different poly-His units (1, 2, 3 and 6 copies of 6-His) were displayed on the seventh loop of the OmpC. Recombinant E. coli strains displaying 1, 3 or 6 copies of poly-His became much more sensitive to SDS (0.1%, w/v) and EDTA (2 mM) compared with control strains. However, recombinant E. coli cells displaying 2 copies of poly-His were resistant to SDS and EDTA; greater than 70% and 90% of cells maintained cell integrity after 60 min treatment with SDS and EDTA, respectively, suggesting its usefulness as a whole cell biosorbent.     

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India

The work was aimed to study the microbial quality of the seafood sold in the domestic markets and incidence of Vibrio parahaemolyticus. Samples comprising of shellfish, finfish, and cephalopods were collected from various fish markets in and around Cochin. Presumed V. parahaemolyticus were identified by standard biochemical tests, and further confirmed by polymerase chain reaction targeting species-specific tl gene (450 bp). About 81% of the samples were found to exceed the limits specified for total plate count while total presumptive V. parahaemolyticus count was above the limit in 71% of the samples ranging from 5.5 × 10⁵ to 9.7 × 10⁷ and 0.31 × 10² to 7.8 × 10⁶ cfu/g, respectively. Pathogenicity of the identified isolates was confirmed by Kanagawa phenomenon and urease activity. A total of 10% of the isolates exhibited weak haemolysis on Wagatsuma agar, and 1% of the isolates showed urease activity using Christensen’s urea agar. Random amplified polymorphic DNA analysis revealed two major clusters based on the species rather than seasonality. The gel pattern revealed 8–10 bands ranging from 0.45 to 3.0 kb. Antibiogram results revealed 85% of the strains sensitive to chloramphenicol and nitrofurantoin. Multiple antibiotic resistance index was found to be 0.4 thus suggesting the risk potential involved in consuming seafoods. The present study has clearly demonstrated the need to adopt seafood safety measures for the products meant for human consumption.     

Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.     

A recombinant vaccine against hydatidosis: production of the antigen in Escherichia coli

A commercial process was developed for producing a recombinant vaccine against hydatidosis in farm animals. The vaccine antigen consisting of a surface protein of the oncospheres of the hydatid worm (Echinococcus granulosus), was produced as inclusion bodies in Escherichia coli. Fed-batch cultures of E. coli using Terrific broth in stirred bioreactors at 37°C, pH 7.0, and a dissolved oxygen level of 30% of air saturation produced the highest volumetric concentrations of the final solubilized antigen. An exponential feeding strategy proved distinctly superior to feeding based on pH-stat and DO-stat methods. The plasmid coding for the antigen was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at 4 h after initiation of the culture. The minimum IPTG concentration for full induction was 0.1 mM.     

Expression and preparation of recombinant hepcidin in Escherichia coli

Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. In this work, hepcidin was expressed in Escherichia coli as a 10.5 kDa fusion protein (His-hepcidin) with a N-terminal hexahistidine tag. The expressed His-hepcidin existed in the form of inclusion bodies and was purified by IMAC under denaturation condition. Since the fusion partner for hepcidin did not contain other cysteine residues, the formation of disulfide bonds was performed before the His-tag was removed. Then, the oxidized His-hepcidin monomer was separated from protein multimers through gel filtration. Following monomer refolding, hepcidin was cleaved from fusion protein by enterokinase and purified with reverse-phase chromatography. The recombinant hepcidin exhibited obvious antibacterial activity against Bacillus subtilis.     

Expression of gloshedobin,a thrombin-like enzyme from the venom of Gloydius shedaoensis,in Escherichia coli

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+). The gene was expressed under T7 promotor with a fusion partner of Thx.Tag and a 6xHis.Tag at its 5 terminal. After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm. Expression at 25°C gave twice the amount of recombinant gloshedobin in cytoplasm than at 37°C.     

Statistical optimization of medium for the production of pyruvate oxidase by the recombinant Escherichia coli

Pyruvate oxidase (PyOD) is a very useful enzyme for clinical diagnostic applications and environmental monitor. Optimization of the fermentation medium for maximization of PyOD constitutively, production by Escherichia coli DH5α/pSMLPyOD was carried out. Response surface methodology (RSM) was used to optimize the medium constituents. A 2^6–2 fractional factorial design (first order model) was carried out to identify the significant effect of medium components towards PyOD production. Statistical analysis of results shows that yeast extract, ammonium sulfate and composite phosphate were significant factors on PyOD production. The optimized values of these three factors were obtained by RSM based on the result of a 2³ central composite rotatable design. Under these proposed optimized medium, the model predicted a PyOD activity of 610 U/L and via experimental rechecking the model, an activity of 670 U/L was attained.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

Relationships between Escherichia coli cells and the surrounding medium during survival processes

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 10(4) cells ml(-1). The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium

In previous studies, we had shown that the buoyant density ofEscherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. the buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting thatE. coli has no mechanisms to sense buoyant density.