Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k_m values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.     

Redox self-sufficient whole cell biotransformation for amination of alcohols

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD⁺ was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP⁺ to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPH-dependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactor-regenerating microorganism with initial addition of 0.005 mM NADP⁺. The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4-trifluorobutyrate with addition of 0.005 mM NADP⁺ three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling.     

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicum l-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67 g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37 g/l 2-methyl-1-butanol and 2.76 g/l 3-methyl-1-butanol in defined medium within 48 h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.     

Resistance of a recombinant Escherichia coli to dehydration

Dehydration of microorganisms, rendering them anhydrobiotic, is often an efficient method for the short and long term conservation of different strain-producers. However, some biotechnologically important recombinant bacterial strains are extremely sensitive to conventional treatment. We describe appropriate conditions during dehydration of the recombinant Escherichia coli strain HB 101 (GAPDH) that can result dry cells having a ∼88% viability on rehydration. The methods entails air-drying after addition of 100 mM trehalose to the cultivation medium or distilled water (for short term incubation).     

Acceleration of azo dye decolorization by using quinone reductase activity of azoreductase and quinone redox mediator

This study demonstrated the effective application of intracellular azoreductase in mediated decolorization of azo dyes. Using the quinone reductase activity of overexpressed azoreductase AZR and quinone redox mediators, the decolorization performance of the recombinant strain Escherichia coli YB was significantly enhanced. In the presence of 0.2 mM lawsone, 75% acid red 27 (1 mM) was decolorized by E. coli YB in only 2 h, which was the highest bacterial decolorization rate ever reported. Compared to lawsone, menadione was a less effective redox mediator. Glucose was found to be the best carbon source for mediated decolorization by E. coli YB. The recombinant strain could complete four rounds of mediated decolorization repeatedly in 12 h. In addition, a 10-min pre-incubation of E. coli JM109 and activated sludge with 2-methylhydroquinone resulted in great improvement of mediated decolorization performance, which may be applied in practical treatment.     

Characterization of a copper-resistant symbiotic bacterium isolated from Medicago lupulina growing in mine tailings

A root nodule bacterium, Sinorhizobium meliloti CCNWSX0020, resistant to 1.4 mM Cu²⁺ was isolated from Medicago lupulina growing in mine tailings. In medium supplied with copper, this bacterium showed cell deformation and aggregation due to precipitation of copper on the cell surface. Genes similar to the copper-resistant genes, pcoR and pcoA from Escherichia coli, were amplified by PCR from a 1.4-Mb megaplasmid. Inoculation with S. meliloti CCNWSX0020 increased the biomass of M. lupulina grown in medium added 0 and 100 mg Cu²⁺ kg⁻¹ by 45.8% and 78.2%, respectively, and increased the copper concentration inside the plant tissues grown in medium supplied with 100 μM Cu²⁺ by 39.3%, demonstrating that it is a prospective symbiotic system for bioremediation purposes.     

Single-step bioconversion for the preparation of l-gulose and l-galactose

Both carbohydrate monomers l-gulose and l-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of l-gulose and l-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing l-galactose at 4.6 g L⁻¹ d⁻¹ and l-gulose at 0.90 g L⁻¹ d⁻¹.     

Production of chiral compound using recombinant Escherichia coli cells co-expressing reductase and glucose dehydrogenase in an ionic liquid/water two phase system

(S)-3-Chloro-1-phenyl-1-propanol ((S)-CPPO) is a useful chiral building block for the synthesis of anti-depressant drugs. The yeast reductase, YOL151W, evidences enantioselective reduction activity, converting 3-chloro-1-phenyl-1-propanone (3-CPP) into (S)-CPPO. Escherichia coli whole cells co-expressing YOL151W and Bacillus subtilis glucose dehydrogenase were employed for the synthesis of CPPO following permeabilization treatment. A reaction system employing these recombinant E. coli whole cells could convert 30 mM 3-CPP enantioselectively into (S)-CPPO. In an effort to enhance substrate solubility and to prevent substrate/product inhibition during the enzyme reaction process, a variety of ionic liquids were tested and [Bmim][NTf₂] was ultimately selected for the ionic liquid/water two phase system. Tween 40 was added to accomplish the efficient mixing of the two phases. Using these recombinant E. coli whole cells and the [Bmim][NTf₂]/water two phase system, 100 mM (S)-CPPO was generated with an enantiomeric excess of >99%.     

Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-l-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-l-fucose resulted in a maximum GDP-l-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer–Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.     

Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli

The recombinant Escherichia coli M15/BCJ2315 which harbored a mandelonitrilase from Burkholderia cenocepacia J2315 was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid. Furthermore, the immobilized cells were reused up to 15 cycles without any activity loss in completely hydrolyzing mandelonitrile (100 mM) within 1 h in aqueous solution. The ethyl acetate–water biphasic system was built and optimized. Under the optimal conditions, as high as 1 M mandelonitrile could be hydrolyzed within 4 h with a final yield and ee value of 99% and 95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for 6 batches, giving a final productivity (g L⁻¹ h⁻¹) and relative production (g g⁻¹) of 40.9 and 38.9, respectively.     

Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli

Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl β-d-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30–34 U/mg for 12 months when stored at 4 °C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.     

Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the host's central carbon metabolism.To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, ¹³C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the ¹³C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol_ATP g_CDW⁻¹ h⁻¹ for the reference strain to 45 mmol_ATP g_CDW⁻¹ h⁻¹ for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5 ± 0.1 mmol g_CDW⁻¹ h⁻¹, hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7 g_CDW mmol_ATP⁻¹ for the reference strain to 4.9 g_CDW mmol_ATP⁻¹ for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of microbial hosts and fermentation conditions.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD⁺-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg^–1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg^–1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg^–1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 mol g^–1 (cell dry weight) min^–1. In the presence of formate, the intracellular [NADH]/[NAD⁺] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.     

Effect of culture conditions on the whole cell activity of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl₂, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD₆₀₀) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.     

Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3 h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12 h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.     

The inhibitor profiling of the caspase family of proteases using substrate-derived peptide glyoxals

A series of substrate-based α-keto-β-aldehyde (glyoxal) sequences have been synthesised and evaluated as inhibitors of the caspase family of cysteine proteases. A number of potent inhibitor sequences have been identified. For example, a palmitic acid containing sequence pal-Tyr-Val-Ala-Asp-glyoxal was demonstrated to be an extremely effective inhibitor of caspase-1, inhibiting not only the action of the protease against synthetic fluorogenic substrates (K_i = 0.3 nM) but also blocking its processing of pro-interleukin-1beta (pro-IL-1β). In addition, the peptide Ac-Asp-Glu-Val-Asp-glyoxal, which is based on the consensus cleavage sequence for caspase-3, is a potent inhibitor of this protease (K_i = 0.26 nM) yet only functions as a comparatively modest inhibitor of caspase-1 (K_i = 451 nM). Potent inhibitor sequences were also identified for caspases-6 and -8. However, the degree of discrimination between the family members is limited. The ability of Ac-Asp-Glu-Val-Asp-glyoxal to block caspase-3 like activity in whole cells and to delay the development of apoptosis was assessed. When tested against caspase-3 like activity in cell lysates, Ac-Asp-Glu-Val-Asp-glyoxal displayed effective inhibition similar to that observed against recombinant caspase-3. Treatment of whole cells with this potent caspase-3 inhibitor was however, not sufficient to significantly stall the development of apoptosis in-vitro.