Showing papers in "Biotechnology and Applied Biochemistry in 2002"

Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2.

Abstract: A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extracellular chitinase was purified to >90% homogeneity from the culture filtrate. The purification involved hydrophobic-interaction and gel-filtration chromatographic separations with a yield of 58%. The purified enzyme (ChiNCTU2) is a monomeric protein with an estimated molecular mass of 36.5 kDa and a pI of 6.3. It is thermally stable at 60 degrees C and pH 6-8 for more than 3 h. The optimal activity is in the range of 50-60 degrees C at pH 7.0. Chitobiose is the predominant product throughout the enzymic hydrolysis of the colloidal chitin, indicating that the purified chitinase is an exo-chitinase. Chito-oligosaccharides [with degree of polymerization (DP) values of 4-6] are good substrates of the purified enzyme, whereas a DP3 oligomer was slowly hydrolysed to form DP1 and DP2 sugars. The first 15 N-terminal amino acids of the enzyme were determined to be ANNLGSKLLVGYWHN, which is highly homologous to that of ChiA from Bacillus cereus. A PCR cloning technique was employed to obtain the corresponding gene from Bacillus NCTU2. The gene sequence was determined to be 1080 bp, encoding a polypeptide of 360 amino acids with the first 27 amino acids as the signal peptide.

Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli.

Abstract: Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

Immobilized lipase‐catalysed production of alkyl esters of restaurant grease as biodiesel

Abstract: Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l. and gran- C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30). All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system. Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran- T.l. and gran- C.a. lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.

Immobilization, stability and esterification studies of a lipase from a Bacillus sp.

Abstract: In the present paper a comparative account of the immobilization of a Bacillus lipase on different solid supports with different surface properties and their thermostability is presented. Immobilization enhanced the thermostability of lipase. At higher temperatures, lipase immobilized and cross-linked on a hydrophobic surface showed the maximum thermostability. The optimum temperature for immobilized lipase was 9 degrees C higher than for the free enzyme, while the pH optima were the same. The half-life of soluble lipase at 50 degrees C was calculated to be 4.5 h, while immobilized lipase did not lose any activity even after 8 h. The temperature stability (for 1 h) of immobilized enzyme was enhanced from 50 to 60 degrees C in comparison with non-immobilized enzyme. Applications of immobilized lipase for esterification are also presented.

Purification and characterization of alkaline protease from Alcaligenes faecalis.

Abstract: Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion-exchange and size-exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 micromol of tyrosine/min per mg of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at 55 degrees C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a K(m) of 1.66 mg/ml and a V(max) of 526 units/min per mg of protein with casein as the substrate.

A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy-root cultures: inactivation by hydrogen peroxide and application in diagnostic kits

Abstract: We have purified various peroxidase isoenzymes from roots and hairy-root cultures of turnip (Brassica napus) which could potentially be used for commercial applications such as an enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation. One of them, a basic peroxidase called HR2, was secreted into the medium of turnip hairy-root cultures. HR2 had a pI of 9.6, a molecular mass of 39.3 kDa and showed great thermostability. The inactivation of HR2 by H2O2 in the absence of reductant substrates was studied. Under these conditions H2O2 acted as a suicide substrate. The kinetic constants calculated have been compared with those of a basic isoperoxidase from horseradish (Armoracia sp.) roots (HRP-C), which is commonly used in commercial kits. The results for HR2 indicated that it was more resistant to inactivation because it presented a lower inactivation efficiency and a higher value for the partition ratio (r=1250) than those described for HRP-C. These results make turnip peroxidase HR2 suitable for use in systems in which high H2O2 concentrations are found. Such an application is demonstrated, namely an enzymic diagnostic kit for determination of uric acid in which HR2 was found to be as efficient as the enzyme originally included in standard kits.

Cashew-tree (Anacardium occidentale L.) exudate gum: a novel bioligand tool

Abstract: The potential of bioaffinity as a tool for the study of biological-recognition mechanisms is gaining increasing value. The search continues for alternative products that can be obtained from renewable sources, such as the bark exudate gum from the cashew tree (Anacardium occidentale L.), which grows wild in many tropical and subtropical countries. Its potential use as a chromatographic matrix and/or for bioaffinity ligand for proteins (lectins) has been investigated. The crude gum was cross-linked in order to obtain a kind of chromatographic matrix (gel). To evaluate the gum's ability to retain glycoproteins (lectins), affinity chromatography was performed and, in addition, the reological behaviour of the gum was characterized.

Production and characterization of a thermostable β-galactosidase from Bacillus coagulans RCS3

Abstract: A strain of Bacillus coagulans RCS3 isolated from a hot-water spring produced significant beta-galactosidase activity at 10 days of growth in a flask. While enzyme production was maximum at 50 degrees C, the highest activity was at 65 degrees C, where the half-life was 2 h. A 2 degrees C decrease in temperature increased the half-life to 15 h without significantly changing the activity, suggesting that 63 degrees C is the temperature of preference compared with 65 degrees C for a combination of good activity and stability. The beta-galactosidase was also stable over pH 5-8, with peak activity at pH 6-7. It was strongly and competitively inhibited by the hydrolysis product galactose. Bivalent cations (Cu(2+), Ni(2+) and Hg(2+)) in the concentration range of 0.5-2.0 mM also inhibited enzyme activity. Both lactose solution and whey could be hydrolysed substantially within 36 h at 50 degrees C. The thermostability and pH-stability and good hydrolytic capability make this enzyme potentially useful in the dairy industry.

Cultivation of Acetobacter xylinum for bacterial cellulose production in a modified airlift reactor.

Abstract: Acetobacter xylinum for bacterial cellulose production was cultivated in a modified airlift reactor. Better results were obtained from the modified reactor than from a conventional bubble column. After 72 h of cultivation, the final concentration of bacterial cellulose was 7.72 g/l and the productivity was 0.107 g/l per h in the modified airlift reactor. The concentration of bacterial cellulose was about three times higher than that produced in the conventional bubble column. Moreover, the bacterial cellulose produced using the modified reactor formed a unique elliptical pellet (the average diameter was 10 mm), which is different from the fibrous form produced using the stirred-tank reactor. The modified airlift reactor with the suspended bacterial cellulose in pellet form had a higher volumetric oxygen-transfer coefficient and mixing capability than that with bacterial cellulose in fibrous form. The dissolved oxygen in the modified airlift reactor could be maintained above 35% throughout the cultivation.

A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger.

Abstract: A comprehensive study on purification and characterization of the two endopolygalacturonases from Aspergillus niger, PG II and PG IV, accounting for 70% of the total polygalacturonase activity, is reported. These enzymes were purified to homogeneity using ion-exchange chromatography and gel filtration. The enzymes had specific activities of 982 and 3750 units/mg, and their molecular masses were 61 and 38 kDa, respectively. The pH optimum of PG II was pH 3.8-4.3 and for PG IV it was between pH 3 and 4.6, and the temperature optima also differed for the enzymes. The enzymes preferred pectic acid as a substrate, cleaving it at random, leading to the release of oligogalacturonides as products. The K(m) values of the two enzymes were found to be 0.12 and 0.72% respectively. The enzymes were rich in hydrophilic amino acids and relatively low in the sulphur-containing amino acids. Both enzymes were rich in beta-structure and differed in their tertiary folding. The tryptophan residues were in a hydrophobic environment. The enzymes differed in their thermal stability; the midpoint of thermal inactivation, T(m), of the two enzymes was found to be 43 degrees C for PG II and 46 degrees C for PG IV.

Effect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures

Abstract: Factors affecting antibody accumulation and stability were investigated in transgenic plant cell cultures. Whereas IgG(1) antibody was stably maintained in media used for animal cell culture, there was a rapid loss over a period of 1-2 h of antibody added to sterile plant culture media. Antibody stability in Gamborg's B5 medium was improved in the absence of Mn. Tobacco suspensions producing IgG(1) antibody were used to test various medium-based strategies for improving antibody accumulation in plant culture. Even though growth was suppressed, antibody levels in the biomass and medium were increased in media containing mannitol at osmolalities up to approximately 450 mOsm x (kg of water)(-1). Adding gibberellic acid and haemin to the cultures was also beneficial, but the effects were not as great as those obtained under hyperosmolar conditions. Moderate increases in antibody accumulation were found by culturing plant cells in B5 medium without Mn.

Application of statistically based experimental designs for the optimization of exo‐polysaccharide production by Cordyceps militaris NG3

Abstract: Statistically based experimental designs were applied to the optimization of medium composition for exopolysaccharide production by Cordyceps militaris NG3 in shake-flask cultures. First, the Plackett‐Burman design was used to search for the main factors on mycelia and exo-polysaccharide production. Among these variables, sucrose and corn steep powder were found to be two significant factors and had positive effects on mycelial yield (with confidence level " 80%) and exo-polysaccharide production (with confidence level " 90%). Subsequently, to study the mutual interactions between variables, the effects of the two main factors on exo-polysaccharide production were further investigated using a central composite design. The optimal composition was found to be 1.03 g/l corn steep powder, 2.95 g/l sucrose, 0.1 g/l K2HPO4, 0.5 g/l MgSO4 [ 5H2O and 0.1 g/l KNO3 for the enhanced production of the exo-polysaccharide, which was 2.604 g/l in shake-flask cultures. Under optimal culture conditions, the maximum exo-polysaccharide concentration in a 5 l stirredtank bioreactor was 3.8 g/l.

Modification of a reactive cysteine explains differences between rasburicase and Uricozyme®, a natural Aspergillus flavus uricase

Abstract: Urate oxidase is used in humans for the control of uric acid in patients receiving chemotherapy. Rasburicase (Fasturtec/Elitek), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, was compared with Uricozyme, the natural enzyme produced by Aspergillus flavus. Rasburicase has a higher purity as demonstrated by SDS/PAGE and chromatographic analysis and a better specific activity. The differences observed for Uricozyme are likely attributable to the previously used purification process, which modifies the enzyme. The production process of rasburicase, on the other hand, preserves the structure of the molecule. MS analysis shows that Uricozyme contains a cysteine adduct on Cys(103). In the crystal structure, the sulphur atom of the cysteine residue in position 103 is orientated to the external surface of the tetramer, whereas the sulphur atom of two other cysteine residues (Cys(35) and Cys(290)) is orientated to the centre of the canal formed by the tetramer. The same adduct is produced by simple incubation of the rasburicase with cysteine.

Production of bacteriocins from Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 using mussel-processing wastes

Abstract: The growth and bacteriocin production by Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 were investigated on mussel-processing wastes. Both bacteriocin productions were satisfactorily modelled using a modified form of the Luedeking and Piret expression, which includes a term for the influence of the pH reduction rate. Experimental data from cultures buffered at different initial concentrations (0, 0.03, 0.10 and 0.25 M) of both bacteria were used to fit and verify the model. The influence of total sugars, nitrogen, phosphorus and buffer concentration on nisin and pediocin production was also studied using response-surface methodology and empirical modelling. Enhanced nisin production (33 BU/ml) was achieved in media buffered with 0.10 M potassium hydrogen phthalate/NaOH. However, the highest levels of pediocin (368 BU/ml) were obtained in the non-buffered media.

High-level expression of human thyroid-stimulating hormone in Chinese hamster ovary cells by co-transfection of dicistronic expression vectors followed by a dual-marker amplification strategy

Abstract: The utilization of dicistronic mRNA expression vectors, containing the gene of interest upstream of an amplifiable marker gene, has shown success in rapidly, efficiently and reproducibly obtaining stable cell lines that express high levels of the protein of interest. For this reason, human thyroid-stimulating hormone (hTSH), a heterodimeric glycoprotein composed of non-covalently linked alpha- and beta-subunits, was expressed in Chinese hamster ovary (CHO) cells using a system based on dicistronic expression vectors. These contained the genes of interest and the amplifiable gene markers dihydrofolate reductase (DHFR) and adenosine deaminase (ADA), separated by an internal ribosome entry site isolated from the encephalomyocarditis virus. After the cells (CHO-DHFR-) had been co-transfected with the expression vectors and submitted to gene amplification in culture medium containing stepwise increments of methotrexate, it was possible to isolate clones that presented a secretion level of up to 7.2+/-1.3 microg/10(6) cells per day, the highest ever reported for the expression of this glycoprotein hormone. A second treatment, involving the utilization of deoxycoformycin, directed to amplify the ADA marker gene, provided a clone with an additional 2-3-fold increase in hTSH secretion, reaching a secretion level of 17.8+/-7.6 microg/10(6) cells per day. Cell culture and hTSH production in a hollow-fibre bioreactor were set up in order to carry out a preliminary physico-chemical, immunological and biological characterization of this hormone in comparison with pituitary-extracted hTSH (from the National Institute of Diabetes and Digestive and Kidney Diseases) and the only recombinant hTSH now available (Thyrogen). The availability of recombinant hTSH is very important in the diagnosis and therapy of thyroid carcinoma, via stimulation of radioiodine uptake.

One-step affinity purification of fetuin from fetal bovine serum.

Abstract: Fetuin is a plasma glycoprotein widely distributed in mammals. It has been used as a model protein for structural analyses and investigations into the biological properties of glycoproteins. A convenient one-step procedure for biospecific isolation of fetuin from fetal bovine serum was developed on the basis of wheatgerm agglutinin (WGA) affinity separation. Two different porous supports, a silica-based material and a polymer-based material, were used for the immobilization of WGA. The prepared WGA adsorbents were characterized and process parameters of the affinity separation of fetuin were investigated and optimized. WGA was immobilized on silica and polymer supports with coupling yields of 99.6 and 99.4% respectively and amounts of coupled ligand of 7.9 and 9.2 mg of WGA/ml respectively. It has been shown that the specific capacities for fetuin were 5.1 mg/ml on WGA-silica, 1.8 mg/ml on WGA-polymer and 4.1 mg/ml on WGA-agarose. All three adsorbents proved to be suitable for the biospecific separation of fetuin. The polymer-based WGA adsorbent was successfully applied in the purification of fetuin from fetal bovine serum in a one-step separation process. The identity and purity of the isolated product was verified by SDS/PAGE. Under optimized conditions up to 21.6 mg of fetuin could be isolated from 1 ml of serum. The procedure described was designed to be easily scaled-up for the production of fetuin.

Determination of total cholesterol in serum by cholesterol esterase and cholesterol oxidase immobilized and co-immobilized on to arylamine glass

Abstract: Commercial cholesterol esterase from bovine pancreas and cholesterol oxidase from Brevibacterium recombinant type have been immobilized individually and co-immobilized on to arylamine glass beads (pore diameter, 55 nm) through diazotization. A method for discrete analysis of total cholesterol in serum was developed employing individually immobilized cholesterol esterase (0.36 mg/50 mg of glass beads) and cholesterol oxidase (0.41 mg/50 mg of glass beads) or co-immobilized cholesterol esterase and cholesterol oxidase (0.56 mg/100 mg of glass beads). Peroxidase from horseradish immobilized on to arylamine glass (0.9 mg/50 mg of glass beads) was common in both the cases. 4-Aminophenazone (0.25 mg/1.5 ml of reaction mixture) and phenol (0.5 mg/1.5 ml of reaction mixture) were used to form dye. In the method, cholesterol ester is hydrolysed by cholesterol esterase to free fatty acid and cholesterol, which is oxidized by cholesterol oxidase to cholestenone and H(2)O(2) x H(2)O(2) is determined enzymically with horseradish peroxidase by additive coupling of 4-aminophenazone with phenol, and the resulting quinoneimine dye is measured at 520 nm, (epsilon=4.0 x 10(-4)). The lower detection limit of the method was 42.8 mg/l for individually immobilized enzymes and 21.4 mg/l for co-immobilized enzymes. Within-day and between-day coefficients of variation were <1.5% and <4.0% respectively for individually immobilized enzymes and <1.0% and <2.5% respectively for co-immobilized enzymes. A good correlation (r=0.99) was found between total serum cholesterol obtained by the present method and a commercial enzo-kit method employing free enzymes. The individually immobilized/co-immobilized enzymes did not show much loss of activity after their 300 uses, when stored at 4 degrees C in distilled water. The co-immobilized enzymes showed better efficiency in terms of sensitivity, linearity and precision compared with individually immobilized enzymes.

Quantitative physical characterization of lipid-polycation-DNA lipopolyplexes.

Abstract: Quantitative assays for the characterization of multi-component lipopolyplexes and their individual constituents are crucial for determining the consistency of formulation protocols which are ultimately reflected in biological activity. Lipid-polycation-DNA formulations consisting of lipids, polycations and DNA are of interest because they have been demonstrated to be efficient gene-delivery vehicles when administered systemically. We have developed a panel of analytical techniques to characterize these lipopolyplexes. Complexes were measured for size by dynamic light scattering and surface-charge characteristics by zeta potential. Interaction between DNA and the polycation, protamine sulphate, was determined using a PicoGreen dye-exclusion technique. Total DNA in the lipopolyplex was assayed through decomplexation of the formulation by addition of heparin sulphate and subsequent DNA quantification by PicoGreen reagent. Protamine sulphate in the lipopolyplex was determined using a novel Amido Black-staining protocol which is linearly sensitive in a range of 0.25-3 microg of protein. Lipids were quantified by HPLC after extraction in chloroform/methanol (2:1). In this method elution is conducted over 40 min, with 1,2-dioleoyl-3-trimethylammonium-propane and cholesterol being resolved by greater than 10 min. Such assays are essential for product characterization and release tests, as well as development of a better understanding of the correlation between physical structure and biological function.

Bioaffinity immobilization of tannase from Aspergillus niger on concanavalin A–Sepharose CL‐4B

Abstract: Tannase from Aspergillus niger van Teighem was immobilized on concanavalin A-Sepharose via bioaffinity interaction. The immobilized enzyme showed a pH optimum similar to that of the free enzyme. K(m) values for free and immobilized enzyme were 0.3 and 0.6 mM respectively. V(max) changed from 0.013 to 0.02 micromol x min(-1) upon immobilization. The immobilized preparation was quite stable to reuse, there was no loss of enzyme activity after three cycles and it retained 81% activity even after the sixth cycle. Ester hydrolysis using the immobilized enzyme led to a 40% conversion into gallic acid as compared with 30% obtained with the free enzyme.

Development of an optimized, simple chemically defined medium for bacterial cellulose production by Acetobacter sp. A9 in shaking cultures.

Abstract: The genus Acetobacter can synthesize cellulose when grown in an undefined medium containing glucose. By using the technique of the omission of a single medium component, an optimized and simple chemically defined medium was developed to support cellulose production by Acetobacter sp. A9 in shaking culture. It contained 4.0% (w/v) glucose, 0.2% (w/v) (NH(4))(2)SO(4), 0.25% (w/v) KH(2)PO(4), 0.3% (w/v) Na(2)HPO(4).12H(2)O, 0.05% (w/v) MgSO(4).7H(2)O, 0.0002% (w/v) FeSO(4).7H(2)O, 0.00025% (w/v) H(3)BO(3), 0.00006% (w/v) nicotinamide, 0.00025% (w/v) inositol and 1.4% (v/v) ethanol. A maximum cellulose concentration of around 8 g/l was achieved after 9 days of cultivation at 200 rev./min. The production of cellulose by Acetobacter sp. A9 was greater in simplified synthetic medium than in complex medium (Hestrin and Schramm medium) conventionally used for Acetobacter strains.

Evaluation of meningococcal C oligosaccharide conjugate vaccines by size-exclusion chromatography/multi-angle laser light scattering.

Abstract: The mean molecular masses of three different meningococcal C saccharide (MenC)-protein conjugate vaccines and their constituent proteins were estimated using HPLC size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and refractive-index (RI) detection (SEC/MALLS). Chromatography of two CRM(197) conjugates (MenC-CRM(197)-A and MenC-CRM(197)-B) and one tetanus toxoid (TT) conjugate (MenC-TT) was performed in PBS, pH 7.4, on TSK-Gel (TosoHaas) analytical columns [CRM(197) is a non-catalytic cross-reacting mutant (CRM) of diphtheria toxin]. Analysis of the light-scattering signal measured at 18 angles simultaneously, using the RI signal as a measure of concentration, gave absolute weight-average-molecular-mass (M(w)) values for the CRM(197) conjugates as follows: MenC-CRM(197)-A, approximately 75,000 g x mol(-1) and MenC-CRM(197)-B, approximately 350,000 g x mol(-1), suggesting that MenC-CRM(197)-A is a monomer (one carrier protein per conjugate molecule), while MenC-CRM(197)-B is largely composed of conjugates containing three or four CRM(197) molecules. The MenC-TT conjugate eluted as a two-component system with (M(w)) of 1.63 x 10(6) and 395,000 g x mol(-1), suggesting that some cross-linked complexes contain up to six TT molecules. Comparison of results from MALLS/RI with those obtained using UV detection highlights the differences in size and relative composition of the various subpopulations of the MenC conjugates that can be obtained using different detection systems.

An adventure in biotechnology: the development of haemophilia A therapeutics -- from whole-blood transfusion to recombinant DNA to gene therapy.

Abstract: The past decade has seen an explosion in the number of therapeutic proteins available for a wide spectrum of diseases. Some of these proteins are obtained from human plasma. Examples of these therapeutic proteins are albumin, intravenous immunoglobulins and prothrombin complex concentrates. The majority of new therapeutic proteins are, however, derived via recombinant DNA technology. There are other examples where the first therapeutic preparation was a crude preparation derived from plasma or tissue and where subsequent development has resulted in a recombinant form of the therapeutic protein. This article focuses on the development of therapeutics for the treatment of haemophilia A (deficiency of Factor VIII activity). The progression from crude plasma fractions to monoclonal-purified preparations to the more recent development of therapeutic concentrates via recombinant DNA technology is described in some detail. Finally, the current status of gene therapy for haemophilia A is evaluated. Both technical issues as well as market forces are described, as both have had significant impact on the product-development process.

Kinetic study of the appearance of an anti-bacterial peptide in the course of bovine haemoglobin peptic hydrolysis.

Abstract: The kinetics of the alpha (1-23) peptide, which is the first anti-bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 degrees C in an homogeneous-phase system. A one-step reversed-phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti-bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin-denaturing agent. Two different mechanisms, 'one-by-one' for native haemoglobin hydrolysis and 'zipper' for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one-by-one or zipper, the anti-bacterial alpha (1-23) peptide is a transient peptide. To prepare the alpha (1-23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DH(c)) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of alpha (1-23) peptide with respect to haemoglobin at the optimal DH(c) values were 55 and 25% respectively.

Characterization and physicochemical properties of a lipase from Pseudomonas mendocina 3121-1.

Abstract: The lipase from Pseudomonas mendocina 3121-1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE. It is composed of two identical subunits. A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW-55F column. Some physicochemical properties of the lipase were investigated using p-nitrophenyl butyrate (p-NPB), Tween 80 solution and Sigma olive-oil emulsion as substrates. The optimum temperature was determined to be 52 degrees C with p-NPB, in the range 50-60 degrees C with Tween 80 and in the range 50-65 degrees C with olive-oil emulsion. The optimum pH was determined to be in the pH range 7.2-7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p-NPB. The enzyme was activated for p-NPB hydrolysis by thermal treatment up to 60 min at 60 degrees C, pH 7.0-8.2, but was rapidly inactivated at 70-80 degrees C and at pH 7.0. The lipase was shown to be more thermolabile at 60 degrees C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 degrees C. The enzyme was found to be pH-tolerant when stored at 20 degrees C, pH 6.3-10.3 (100 mM Briton-Robson buffer) as the half-life (t(1/2)) was more than 240 h when p-NPB was used as the substrate. By contrast, the pH-stability range was more narrow (pH 8.0-10.5) with olive-oil emulsion. The effect of various metal ions and EDTA depended on the nature of the substrate.

Purification and biochemical characterization of the carbamate hydrolase from Pseudomonas sp. 50432

Abstract: A soluble carbamate hydrolase that had a wide specificity was purified 2032-fold from Pseudomonas sp. 50432. This was achieved using a combination of anion-exchange, gel-filtration and hydrophobic-interaction- chromatography techniques. Carbamate hydrolase cleaved the ester linkage of the N-methylcarbamates. The native enzyme was a monomer with a molecular mass of 88 kDa. The optimum pH and temperature of the enzyme activity were 8.5 and 37 degrees C respectively. The tested cations or EDTA did not affect the enzyme activity. However, 2-mercaptoethanol reversibly inhibited the enzyme activity. The enzyme showed the K(m) values of 16 and 12 microM for carbofuran and carbaryl respectively. The purified enzyme did not hydrolyse o-nitrophenyl dimethylcarbamate but hydrolysed several N-methylcarbamates and 1-naphthyl acetate.

The effect of chitin and chitosan on fibroblast‐populated collagen lattice contraction

Abstract: The effects of chitin [(1-->4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the human dermal fibroblast-mediated contraction of collagen lattices were examined in vitro as a model for the contraction of cutaneous wounds in vivo. Chitosan CL313A, a short-chain-length 89% deacetylated chitosan chloride, inhibited fibroblast-populated collagen lattice (FPCL) contraction at higher initial concentrations (500 and 1,000 microg/ml) in FPCLs fabricated with responsive dermal fibroblasts, while in FPCLs containing non-responsive fibroblasts inhibition of contraction was reduced. The responsive and non-responsive phenotype of human dermal fibroblasts to treatment with chitosan CL313A has been reported previously by us. The inhibition of fibroblast-mediated collagen lattice contraction by chitosan appeared to be strongly correlated with whether the cells were responsive or non-responsive. The effect of chitin-50A on fibroblast-mediated collagen lattice contraction was also examined to investigate whether the level of deacetylation was important for its inhibitory effect on contraction. However, this had no effect on contraction at the concentrations tested, supporting previous work that only chitosan samples with higher levels of deacetylation showed any biological activity. This work indicates that highly deacetylated chitosan inhibits fibroblast-mediated contraction of collagen lattices and may therefore be useful as a therapeutic agent to reduce contraction and therefore scarring in wound healing in vivo.

Enhancement of the immune response generated against hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids.

Abstract: Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti-E2 antibodies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon-gamma enzyme-linked immunospot (ELISPOT) assay, and induced detectable levels of interferon-gamma but not interleukin-4 in vaccinated mice. The detection of both antibody and cytotoxic T-lymphocyte responses, potentially targeted to circulating or cell-infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.

Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose.

Abstract: Multi-substrate specificity of neopullulanase towards cyclodextrin, acarbose and maltose was investigated using a clone originating from Bacillus stearothermophilus IMA6503. The enzyme purified from Escherichia coli harbouring the corresponding nplA gene hydrolysed beta-cyclodextrin (beta-CD) to maltose and glucose. It exhibited substrate preference for beta-CD, starch and pullulan in the proportions of 10.4:1.2:1. The enzyme not only hydrolysed acarbose, an alpha-amylase inhibitor, to a pseudotrisaccharide (PTS) and glucose, but also transferred PTS to glucose, forming isoacarbose. Moreover, it hydrolysed maltose to glucose and transferred the glucose to another maltose molecule to form panose when maltose was present at a low concentration (0.5%) in the reaction solution. The enzyme catalysed condensation between two maltose molecules and subsequent hydrolysis of the resulting 6(2)-O-alpha-maltosyl-maltose to glucose and panose, when maltose concentration was increased to 20%. Neopullulanase was likely to be present in monomer-dimer equilibrium with a molar ratio of 1:9 in 50 mM sodium acetate buffer (pH 6.0). The association-dissociation equilibrium of neopullulanase was shifted to monomerization by KCl. When the content of monomer increased in the reaction mixture, the specific activity towards soluble starch increased to 150%, while that towards beta-CD decreased to 80%. Therefore, multi-substrate specificity of neopullulanase was likely to be modulated by the shift of monomer-dimer association equilibrium.

An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions.

Abstract: The Escherichia coli outer-membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2'. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon-like peptide-1 (7-37) in 4 M urea. The P1-P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The k(cat)/ K(m) ratio increased with basic residues at P4 or P6, mainly due to a lower K(m) rather than an increase in k(cat). On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli-expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.

Production of non-alcoholic beer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle.

Abstract: Production of non-alcoholic beer using Saccharomyces cerevisiae has been studied. Non-recombinant mutant strains with a defect in the synthesis of tricarboxylic-acid-cycle enzymes were used and applied in both free and pectate-immobilized form, using both batch and packed-bed continuous systems. After fermentation, basic parameters of the beer produced by five mutant strains were compared with a standard strain of brewing yeast. Results showed that the beer prepared by mutant yeast cells was characterized by lower levels of total alcohols, with ethanol concentrations between 0.07 and 0.31% (w/w). The organic acids produced, especially lactic acid, in concentrations up to 1.38 g x l(-1) had a strong protective effect on the microbial stability of the final product and thus the usual addition of lactic acid could be omitted. Application of the yeast mutants appears to be a good alternative to the classical methods for the production of non-alcoholic beer.