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The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.

The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule. The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p. In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology. The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments. Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively. Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures. The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides. The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.

The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator. To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis. In this paper, Phe119 and Leu167, which are located in the acyl portion of the substrate-binding pocket of the lipase of B.cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis. The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation. The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate, p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates. Two mutant lipases, L167V and F119A/L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro. It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester.

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene ( phaZ ) was identified near the PHA synthase gene ( phaC ) of P. denitrificans . Cell extract of Escherichia coli carrying lacZ–phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D (−)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg 2+ ion inhibited PHB degradation. However, the inhibitory effect by Mg 2+ ion was not observed using the cell extract of P. denitrificans .

Phospholipase D (PLD), secreted into the culture medium of an actinomycete, Streptoverticillium cinnamoneum, has been purified to homogeneity and characterized. The Stv. cinnamoneum PLD efficiently catalyzes both the hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially Al3+. Nucleotide and amino acid sequence determination of the Stv. cinnamoneum PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.

Several alcohols were examined as substrates for the polyhydroxyalkanoate synthesis by Paracoccus denitrificans. The bacterium synthesized a homopolyester of poly(3-hydroxybutyrate) from ethanol. When n-pentanol was used as growth substrate, homopolyester poly(3-hydroxyvalerate) was synthesized, whereas copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulated during bacterial growth on n-propanol. When alcohols were automatically fed as growth substrates, ethanol, n-propanol, and n-pentanol gave higher polyester content. Although poly(3-hydroxybutyrate) was synthesized from methanol or n-butanol, its content was very low. Under nitrogen-deficient conditions, polyester;content in cells increased, especially with ethanol, n-propanol, and n-pentanol. Using a mixture of two alcohols P. denitrificans could synthesize polyesters with varying relative ratios of 3-hydroxybutyrate to 3-hydroxyvalerate.

The beta-ketothiolase gene (phaA) and acetoacetyl-CoA reductase gene (phaB) were isolated from Paracoccus denitrificans. Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40,744 Da and of 242 amino acids with a molecular mass of 25,614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4-74.0% for the phaA gene product and 47.6-80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli. In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA-phaB, in P. denitrificans. NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.

Theoretical yields of poly-d(−)-3-hydroxybutyrate (PHB) from several carbon sources have been estimated from biochemical pathways leading to PHB. In estimating the yields, a special emphasis is made on recycling (or regeneration) of NADP+ which is the co-substrate of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADP+-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. Theoretical and observed yields have been compared when polyhydroxyalkanoates (PHA) were synthesized from methanol and from n -amyl alcohol by a methylotroph, Paracoccus denitrificans . An equation, which predicts the overall yield of PHB when allowance is made for non-PHB biomass formation in actual bacterial PHB production, has been derived as a function of both theoretical yields and PHB content of the total dry cell mass. The ratio of the overall (yield) to be theoretical yield is roughly proportional to the PHB content. A novel specific PHB formation rate on the basis of the residual biomass (total biomass-PHB contained) was proposed. The specific PHB formation rate decreased according to a mono-molecular decay model whose decay constant depended solely on the C/N ratio of the feed solution. From this model, an equation has been derived to calculate the volumetric productivity of PHB on the assumption that the total amount of the residual biomass is unchanged in the nitrogen-deficient PHB formation phase. Also, a graphical procedure has been shown to calculate the volumetric productivity of PHB. A comparison has been made between several data of PHB productivities that have been calculated from the literature.

n-Amyl alcohol was examined as a source for the synthesis of the 3-hydroxyvalerate (3HV) unit of the biopolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), by Alcaligenes sp., Pseudomonas sp. and several methylotrophic bacteria. A. eutrophus and Ps. lemoignei synthesized P(3HB-co-3HV) from glucose and n-amyl alcohol under nitrogen-deficient conditions. Many of methylotrophic bacteria grown on methanol synthesized the copolyester from methanol and n-amyl alcohol under nitrogen-deficient conditions. The content and composition of the polyester varied from strain to strain. Paracoccus denitrificans differed from all others in having a higher content of 3-hydroxyvalerate units in the copolyester synthesized.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.

Six lipases were examined for concentrating the n-3 polyunsaturated fatty acid (n-3 PUFA) of two kinds of fish oil (cod liver oil and refined sardine oil). Although all lipases could increase the n-3 PUFA content of the remaining glycerides, Candida cylindracea and Aspergillus niger lipases gave glycerides with a more than two-fold increase in n-3 PUFA content over the original fish oils.Candida cylindracea lipase seems the most promising with respect to recovery of triacylglycerol. Aspergillus niger lipase increased not only the docosahexaenoic acid (DHA) content but also eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA), although the absolute value of the latter was quite low. The effects of temperature (15–40 °C) on the concentration of n-3 PUFA were investigated. Lower temperatures did not improve the concentration of n-3 PUFA, but prevented the development of an unpleasant odor in the product.

Olive oil was hydrolyzed continuously at 40oC by Candida cylindracea lipase in a small hollow fiber bioreactor (total area of hollow fibers was about 0.11 m2) in which the hollow fibers were made of microporous polypropylene. The lipase could be adsorbed easily onto oil-impregnated hollow fibers from its aqueous solution. The continuous feedings of olive oil inside the hollow fibers and of the buffer solution containing 18% glycerol as a stabilizer outside the hollow fibers were started after the enzyme-glycerol solution was removed from the bioreactor and the buffer-glycerol solution was added. An unvaried half life of 14 days of the adsorbed enzyme was observed when increasing amounts of the enzyme (1.0~5.0mg/ml) were put in, but its half-life was lowered to 6 days when the amount of the added enzyme was less (0.05mg/ml). Free enzymes in the enzyme solutions with and without 18% glycerol retained their initial activities equally for at least 3 months at temperatures below 4oC. This suggests the feasibil...

Continuous synthesis of oleic acid glycerides by seven lipases available commercially were compared at 40°C using the microporous hydrophobic membrane bioreactor we developed recently. The lipases produced by Rhizopus japonicus, Candida cylindracea, and Phycomyces nitens were quickly inactivated in 97% glycerol solution and yielded very low glyceride conversions. The other four lipases yielded glyceride synthesis activities in the order: Rhizopus delemer’s

The effects of fat hydroperoxides on the reaction rate and enzyme stability in glycerolysis of safflower oil catalyzed by Pseudomonas fluorescens lipase were investigated. In the batch glycerolysis, the initial reaction rate, ri, decreased linearly with the increase in the safflower oil's peroxide value (POV). In the continuous glycerolysis using a microporous hydrophobic membrane bioreactor, the outlet conversions decreased monomolecularly with the elapsed time due to inactivation. The first-order decay constants, Ka, increased with the increase in the safflower oil's POV. The courses of sodium dodecyl suit ate-pol yacrl lamide gel electrophoresis (PAGE) of the enzyme in the glycerol phase in the continuous glycerolysis showed that the high hydroperoxide content caused polymerization of the lipase. From the activity staining of the bands of the native proteins developed in the PAGE, the highly polymerized lipase was found to have no lipolytic activity.