Syntheses and Biological Activities of Analogs of Abscisic Acid

Selenium dioxide oxidation of methyl α-ionylideneacetate (IIb) in ethanol afforded methyl 1′-and 4′-hydroxy-α-ionylideneacetate (IIIb and IV), methyl 3′-hydroxy-β-ionylideneacetate (V) and crude dihydroxy-ionylideneacetate (VI). The latter was oxidized with active manganese dioxide to give methyl abscisate (Ib). The growth and germination-inhibitory activity of compounds related to abscisic acid on Azuki bean seedlings and some species of seeds were examined.     

Biological Activities of Melanotropic Peptide Fatty Acid Conjugates

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the α-MSH fragment analog, H-Asp⁵-His⁶-D-Phe⁷-Arg⁸-Trp⁹-Lys¹⁰-NH₂. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to α-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a “creeping” potency in the lizard skin bioassay—that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10–100 times more active than α-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Ast⁵,D-Phe⁷,Lys¹⁰]α-MSH₅-₁₀-NH₂, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

Candida Antarctica Lipase B-Catalysed Synthesis Of Dihydroxyacetone Fatty Acid Esters

The enzymatic syntheses of 1-lauroyl-dihydroxyacetone and 1, 3-dilauroyl-dihydroxyacetone were investigated. Lipase B from Candida Antarctica (SP435) was used to catalyse the acylation of dihydroxyacetone (DHA) with lauric acid in organic solvent media at controlled water activity. High conversions of dihydroxyacetone (< 90%) are achieved when the water activity is 0.11 or below in solvents of various hydrophobicities, such as diethyl ether, methyl-terr-butyl ether (MTBE) and diphenyl ether. The main product in the esterification of DHA with lauric acid is 1-lauroyl-DHA, while the amount of 1, 3-dilauroyl-DHA that is produced can be increased by changing the reaction conditions. Thus, hasing the water activity from 0.75 to 0.06 resulted in an increase in the total yield of 1, 3-dilauroyl-DHA from 3% to 20%. Solvents which have high logP values favoured the acylation of 1-lauroyl-DHA and thereby the formation of 1, 3-dilauroyl-DHA. Thus, when diphenyl ether was used in this reaction, the yield of 1, 3-dilauroyl-DHA was 45%. Complete acylation to 1, 3-dilauroyl-DHA was achieved when a fatty acid vinyl ester was used as acyl donor in a closed reactor.     

Syntheses and Biological Activities of Isonitrile Dipeptides

We investigated GroEL substrates from Bacillus subtilis 168 using the single-ring mutant of B. subtilis GroEL. We identified 28 candidates for GroEL substrates, of which Spo0B, Ald, Eno, SpoIIP, and FbaA were involved in spore formation, and Rnc, Tuf, Eno, Tsf, and FbaA were essential for B. subtilis growth. As observed at the protein level, the amount of SpoIIP interaction with GroEL increased at 3 h after initiation of sporulation.     

Multiple phosphorylation of rat liver 3-hydroxy 3-methylglutaryl coenzyme a reductase

Rat liver homogeneous ³²P-labeled hydroxy methylglutaryl coenzyme A reductase, was treated independently with CNBr and trypsin and the resulting [³²P]phosphopeptides were analyzed by disc gel electrophoresis. CNBr treatment produced only one ³²P-fragment of Mr 18,000. The time course of trypsin hydrolysis initially showed the appearance of some phosphopeptides, which were lately converted in two phosphopeptides of low Mr. These results provide direct support for the concept that hydroxy methyl glutaryl coenzyme A reductase kinase solubilized from microsomes phosphorylates only two sites or set of sites in the reductase molecule.     

Syntheses and Biological Activities of New Plant Growth Inhibitors Structurally Related to Abscisic Acid

Several analogs of abscisic acid (ABA) were prepared and their biological activities were assayed. Among the compounds tested, 5-(l, 2-epoxy-2, 6, 6-trimethyl-l-cyclohexyl)-, 5-(l-hydroxy-2, 6, 6-trimethyl-2-cyclohexen-l-yl)- and 5-(l-hydroxy-2-methylene-6, 6-dimethyl-l-cyclohexyl)-3-methyl-cis, trans-2, 4-pentadienoic esters (V, IX, XXIII and XXV) were found to be potent plant growth inhibitors. Their activities were superior or comparable to that of ABA.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Syntheses and Biological Activities of Danicalipin A Derivatives

Synthesis of three derivatives of danicalipin A, tetrachloride, trisulfate and a fluorescent probe was achieved through Wittig reaction strategy. Toxicity of the derivatives against brine shrimp (Artemia salina) as also investigated to provide useful information for the biological activity; i) less chloride derivative showed similar toxicity to danicalipin A, ii) the amphiphilic property, a characteristic feature of danicalipin A, was crucial because trisulfate considerably decreased the toxicity and iii) fluorescent derivative kept brine shrimp toxicity of danicalipin A.     

Syntheses and Biological Activities of Dihydro-5,6-dehydrokawain Derivatives

The syntheses and biological activities of dihydro-5,6-dehydrokawain derivatives against plant pathogenic fungi and termites were investigated. Dihydro-5,6-dehydrokawain was isolated by a simple method without chromatography from the leaves of Alpinia speciosa K. Schum. The white crystalline compound obtained was identified as dihydro-5,6-dehydrokawain (1) by instrumental analyses. 4-Hydroxy-6-(2-phenylethyl)-2H-pyran-2-one (3) was prepared by hydrolyzing dihydro-5,6-dehydrokawain. Three dihydro-5,6-dehydrokawain derivatives were synthesized by reacting 3 with phosphoric agents.

Among the synthesized compounds, dimethyl [6-(2-phenylethyl)-2-oxo-2H-pyran-4-yl] phosphorothionate (4) had the strongest antifungal activity of 91% at 100 ppm against Corticium rolfsii.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

The Use of Fatty Acid Esters to Enhance Free Acid Sophorolipid Synthesis

Fatty acid esters were prepared by transesterification of soy oil with methanol (methyl-soyate, Me-Soy), ethanol (ethyl-soyate, Et-Soy) and propanol (propyl-soyate, Pro-Soy) and used with glycerol as fermentation substrates to enhance production of free-acid sophorolipids (SLs). Fed-batch fermentations of Candida bombicola resulted in SL yields of 46 ± 4 g/l, 42 ± 7 g/l and 18 ± 6 g/l from Me-Soy, Et-Soy, and Pro-Soy, respectively. Liquid chromatography with atmospheric pressure ionization mass spectrometry (LC/API-MS) showed that Me-Soy resulted in 71% open-chain SLs with 59% of those molecules remaining esterified at the carboxyl end of the fatty acids. Et-Soy and Pro-Soy resulted in 43% and 80% open-chain free-acid SLs, respectively (containing linoleic acid and oleic acid as the principal fatty acid species linked to the sophorose sugar at the omega-1 position), with no evidence of residual esterification. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Survival of poly-3-hydroxybutyrate-producing bacteria in soil microcosms

Bacillus megaterium is a potential bioremediation and biocontrol agent. The accumulation of reserve polymers, such as poly-3-hydroxybutyrate (PHB), increases survival of B. megaterium in water. We used wild-type strains of this species and mutant strains deficient in PHB synthesis in soil microcosms for testing the hypothesis that differences in survival capabilities and spore quality between strains is maintained in heterogeneous environments enriched with organic matter. No differences in survival between strains, nor a decrease in bacterial cell numbers were observed in sterile soil microcosms. In non-sterile soil, the total cell number (vegetative cells plus spores) of the PHB wild-type strain was 3.5 times higher than that of the PHB-negative mutant. We suggest that for predictive purposes, validation of survival in a variety of conditions is necessary.     

Phase Behaviour and Formation of Fatty Acid Esters Nanoemulsions Containing Piroxicam

Fatty acid esters are long-chain esters, produced from the reaction of fatty acids and alcohols. They possess potential applications in cosmetic and pharmaceutical formulations due to their excellent wetting behaviour at interfaces and a non-greasy feeling when applied on the skin surfaces. This preliminary work was carried out to construct pseudo-ternary phase diagrams for oleyl laurate, oleyl stearate and oleyl oleate with surfactants and piroxicam. Then, the preparation and optimization study via ‘One-At-A-Time Approach’ were carried out to determine the optimum amount of oil, surfactants and stabilizer using low-energy emulsification method. The results revealed that multi-phase region dominated the three pseudo-ternary phase diagrams. A composition was chosen from each multi-phase region for preparing the nanoemulsions systems containing piroxicam by incorporating a hydrocolloid stabilizer. The results showed that the optimum amount (w/w) of oil for oleyl laurate nanoemulsions was 30 and 20 g (w/w) for oleyl stearate nanoemulsions and oleyl oleate nanoemulsions. For each nanoemulsions system, the amount of mixed surfactants and stabilizer needed for the emulsification to take place was found to be 10 and 0.5 g (w/w), respectively. The emulsification process via high-energy emulsification method successfully produced nano-sized range particles. The nanoemulsions systems passed the centrifugation test and freeze–thaw cycle with no phase failures, and stable for 3 months at various storage temperatures (3°C, 25°C and 45°C). The results proved that the prepared nanoemulsions system cannot be formed spontaneously, and thus, energy input was required to produce nano-sized range particles.     

Lipase-Catalyzed Synthesis of Fatty Acid Esters Useful in the Food Industry

Enzyme reactions are very attractive in food technology because they can be carried out under mild conditions and without toxic solvents and other catalysts. Lipases can esterify various alcohols with fatty acids. There are opportunities to synthesize useful compounds with special functions as food materials by using the catalytic function of lipase. Reverse micellar systems are discussed as reaction systems for lipases in organic solvents, especially in triacylglycerol synthesis using phosphatidylcholine as the surfactant. Syntheses of some amphiphilic substances including O-acyl-L-homoserine are also discussed.     

Lipase-Catalyzed Synthesis of Fatty Acid Esters Useful in the Food Industry

Enzyme reactions are very attractive in food technology because they can be carried out under mild conditions and without toxic solvents and other catalysts. Lipases can esterify various alcohols with fatty acids. There are opportunities to synthesize useful compounds with special functions as food materials by using the catalytic function of lipase. Reverse micellar systems are discussed as reaction systems for lipases in organic solvents, especially in triacylglycerol synthesis using phosphatidylcholine as the surfactant. Syntheses of some amphiphilic substances including O-acyl-L-homoserine are also discussed.     

重组菌Ralstonia eutropha W50-EAB D-木糖代谢相关的限速靶点

【目的】通过代谢工程改造真养罗氏菌(Ralstonia eutropha)W50-EAB木糖代谢的相关限速靶点,进一步提高R.eutropha W50-EAB的D-木糖利用效率,为获得高效利用纤维素水解液的菌株奠定基础。【方法】利用PCR技术扩增R.eutropha转酮酶基因tkt A,cbb T2和转醛酶基因tal,将扩增的tkt A,cbb T2和tal基因分别构建到表达载体p BBR1MCS-3上,获得重组质粒p WL1-TKT,p WL1-CBBT2,p WL1-TAL。通过电转的方式将质粒分别转化W50-EAB获得重组菌W50-KAB,W50-CAB和W50-TAB。利用基因敲除的方法,获得醛还原酶基因h16_A3186敲除株W50’-EAB。通过电转的方式将重组质粒p WL1-TAL导入敲除株W50’-EAB获得重组菌株W50’-TAB。通过摇瓶发酵研究重组菌株W50-KAB,W50-CAB,W50-TAB,W50’-EAB以及W50’-TAB的发酵特性。【结果】酶活分析结果表明,转酮酶和转醛酶基因实现表达。摇瓶发酵结果表明,转酮酶基因过表达菌株W50-KAB和W50-CAB相比于对照菌株W50-EAB/p3,表现出降低的木糖利用能力;而转醛酶基因过表达重组菌株W50-TAB以及敲除菌株W50’-EAB对木糖的利用得到一定的提高。在0.1 mol/L木糖的发酵培养基中,W50-EAB的最大比生长速率为0.035 h-1,PHB干重比为16.2±1.01%;而W50-TAB的最大比生长速率提高到0.039 h-1,PHB干重比达到20.5±0.76%;醛还原酶基因敲除菌株W50’-EAB最大比生长速率提高到0.040 h-1,PHB含量提高到19.8±1.05%。结果显示转醛酶基因的过表达与醛还原酶基因的敲除对木糖利用均表现出一定的优势,将这两种优势组合获得菌株W50’-TAB,摇瓶发酵分析结果为最大比生长速率达到0.042 h-1,PHB积累达到27.9±0.47%,相比于对照菌株提高了72.2%。另外,在含有葡萄糖(0.01 mol/L)和木糖(0.09 mol/L)的混合糖培养下,重组菌株W50-TAB,W50’-EAB和W50’-TAB相比于在纯木糖培养下都表现出更高的生物量和胞内PHB积累量。【结论】磷酸戊糖途径关键酶转醛酶基因的过表达加速了木糖代谢流,从而可以高效利用木糖积累一定量的PHB。醛还原酶对木糖代谢有阻碍作用,敲除该酶基因后木糖代谢能力有了一定的提高,而两者协同作用可以进一步提高重组菌株的木糖利用效率和PHB积累能力。