Ester synthesis from α-substituted carboxylic acid catalyzed by polyethylene glycol-modified lipase fromCandida cylindracea in benzene

Summary Lipase fromCandida cylindracea was coupled with polyethylene glycol(PEG). In contrast to the previously used lipase fromPseudomonas fluorescens, the modified lipase catalyzed the ester synthesis in benzene at 25°C from short-chain alcohols and - or -substituted carboxylic acid. Using the modified lipase, following esters were synthesized; pentyl -methylpentanate, methyl benzoate, and methyl retinoate.     

Synthesis of eicosapentaenoyl phosphatidylcholines by polyethylene glycol-modified lipase in benzene

Summary Lipase fromCandida cylindracea was modified with activated polyethylene glycol, which was synthesized from -carboxymethyl--methoxypoly-(oxyethylene) and N-hydroxysuccinimide with dicyclohexylcarbodiimide. The modified lipase was soluble in organic solvents such as benzene and catalyzed the reaction of ester-exchange to synthesize eicosapentaenoyl phosphatidylcholines from dipalmitoyl phosphatidylcholine and eicosapentaenoic acid. Either one of palmitic acid at C-1 or C-2 position of the phosphatidylcholine was exchanged with eicosapentaenoic acid.     

Solvent production byClostridium pasteurianum in media of high sugar content

Summary The fermentation end products ofClostridium pasteurianum ATCC 6013 are normally acetic and butyric acids. When grown in media of high sugar content however, significant quantities of solvents (acetone, butanol and ethanol) were produced. Solvent production was not stimulated by added acetic and butyric acids, nor was the effect due to a low water activity of the mediumper se.     

Ester synthesis in benzene by polyethylene glycol-modified lipase from Pseudomonas fragi 22.39B

Lipase (EC 3.1.1.3) from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase was soluble in organic solvents such as benzene and chlorinated hydrocarbons, and catalyzed the synthesis of esters from fatty acids and alcohols in these solvents. The longer the chain length of fatty acid, the higher the ester synthesis activity. A similar specificity was not observed with other substrates like alcohol. Values of K_m and V_max were revealed by kinetic study on the ester synthesis reaction with the modified lipase in benzene. Fatty acids with branched carbon chain at the position neighboring the carboxyl group did not serve as substrates of ester synthesis.     

Inhibition of cultured cell growth by tungstate and molybdate

The growth of suspension cultured cells of Nicotiana tabacum (tobacco) was inhibited completely by 100 M tungstate. Even though molybdate reversed the tungstate inactivation of nitrate reductase activity, the growth inhibition was not reversed. The growth inhibition of N. tabacum, Daucus carota, Glycine max and Solanum tuberosum suspension cultured cells by tungstate was similar in media with or without amino acids as a source of reduced nitrogen. Only in the case of G. max was a slight reversal caused by the amino acids. Tungstate was slightly less inhibitory to the growth of a nitrate reductase-lacking mutant N. tabacum line (nia-63) than to the line with nitrate reductase. These results indicate that tungstate must inhibit the cell growth of the four species used, predominantly, in some way other than by inhibiting nitrate reductase activity. Similar studies with molybdate, a sulfate analog which apparently competes with sulfate at the ATP sulfury-lase enzyme, showed that 1 mM concentrations were completely inhibitory to cell growth. The addition of sulfate or cysteine, as a source of reduced sulfur, and amino acids, as a source of reduced nitrogen, in most cases did not reverse the molybdate inhibition appreciably. Some reversal was seen only by sulfate with D. carota cells and by cysteine plus amino acids with D. carota and G. max. These results indicate that selection for tungstate or molybdate resistance will in general not select for higher levels or other alterations in the activity of nitrate reductase or ATP sulfurylase, respectively, since these ions do not inhibit growth by primarily affecting these enzymatic steps in cultured cells of the four species studied.     

Regulation of cell proliferation and morphogenesis by amino acids inBrassica tissue cultures and its correlation with threonine deaminase

The effect of amino acids has been investigated with respect to the capacity ofBrassica cultures to undergo proliferation and differentiation. Hormone medium without any amino acid resulted in 6% shoot formation. Addition of optimal concentrations of L-leucine and L-isoleucine enhanced shoot formation upto 30% and 60%, respectively. L-methionine, L-threonine and pyruvic acid supported only proliferation but no differentiation. Amino acids had a marked effect on the activity of enzyme threonine deaminase (TD), bothin vivo andin vitro. TD in proliferating callus cultures was 3-fold higher than in differentiating cultures. Amino acids which induced cell proliferation increased TD while those which supported differentiation repressed it. Amino acids which did not alter TD activity had no effect on morphogenesis. The results suggest that amino acids play a regulatory role inBrassica morphogenesis which can be correlated with the activity of threonine deaminase.     

Enantioselective oxidation of mandelic acid using a phenylmalonate metabolizing pathway of a soil bacteriumAlcaligenes bronchisepticus KU 1201

Summary The metabolic pathway of phenylmalonic acid byAlcaligenes bronchisepticus KU1201, which catalyzes the asymmetric decarboxylation of -aryl--methylmalonic acids to -arylpropionic acids, is demonstrated. Enantioselective oxidation of mandelic acid was investigated using the metabolizing pathway in this strain. Incubation of (±)-mandelic acid withA. bronchisepticus afforded optically pure (R)-one accompanied by benzoylformic acid and benzoic acid. This enzymatic oxidation is also applicable to various substituted mandelic acids.     

Propagation of Costus speciosus (Koen.) Sm. through in vitro rhizome production

Rhizomes developed in vitro on shoots of Costus speciosus (Koen.) Sm. which were initiated from zygotic embryos. The effect of different hormonal and nutritional additions to Schenk and Hildebrandt' s (SH) basal nutrient medium on rhizome development was studied. Rhizomes developed on SH basal salts but formed with increased frequency on medium supplemented with adenine sulfate, casamino acids (CA) and various combinations of cytokinins and auxins. Incubation in light was necessary for rhizome formation. Isolated basal as well as nodal (aerial) rhizomes formed and produced new shoots. In basal medium without any growth additives (control) the average number of shoots produced per inoculum was 3.3 ±0.73 whereas in the best suited medium i.e. supplemented with 250 mg l^–1 CA the number of shoots obtained was 22.7±1.92. There was no dormancy period for rhizomes under the cultural conditions investigated. Rhizome-sprouts were easily transplanted from the in vitro conditions to the field. More than five hundred propagules (i.e. sprouted-rhizomes) were obtained within 5 months and it has been estimated that more than 2.4 × 10⁵ propagules could be achieved per year through four subsequent in vitro rhizomes-generation cycles. Thus, a rapid method of propagation has been established.Abbreviations AdS Adenine sulphate - BA benzyladenine - CA casamino acids (vitamin free) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - Kn Kinetin - NAA naphthaleneacetic acid - SH Schenk and Hildebrandt's medium (1972)     

The macromolecular composition and essential amino acid profiles of strains of Zymomonas mobilis

Summary From continuous culture studies it has been shown that the protein concentrations of strains of Z. mobilis (62–68%) were appreciably higher than for the yeast S.uvarum (45–50%). The DNA and RNA contents were similar for the two species. Comparison of the essential amino acids indicated that Z.mobilis did not exhibit the deficiency in methionine which was apparent in the yeast. Such a study of the macromolecular composition of cells of Z.mobilis is important in assessing its by-product nutritional value for animal feed supplementation.     

Chemical modification of the beta-subunit isolated from a membrane-bound Fo-F1-ATP synthase: modification by 4-chloro-7-nitrobenzofurazan does not inhibit restoration of ATP synthesis or hydrolysis

The purified, reconstitutively active, β-subunit of the Fo·F₁-ATP synthase of Rhodospirillum rubrum chromatophores was found to bind both 4-chloro-7-nitrobenzofurazan (NBD-Cl) and dicyclohexylcarbodiimide (DCCD). The binding stoichiometry at saturation was 1 mol of either reagent per mol of β. The NBD-modified β-subunit did rebind to the β-less chromatophores and restored all their lost ATP-linked activities as efficiently as the untreated β, whereas the DCCD-modified β-subunit lost completely its capacity to rebind to the depleted chromatophores. These results suggest that the amino acid residue which is modified by NBD-Cl in the isolated β-subunit is not essential for binding and may be also not for activity.     

Synthesis of Analogues of Ribosylbarbituric Acid

Abstract

Syntheses of 5-alkyl (5 b, c), 2′-deoxyribosyl (9, 10) and arabinofuranosyl (13) analogues of ribosylbarbituric acid (5a) are described. The compounds were prepared by condensation of persilylated barbituric acids with pentofuranosyl moieties. The 2′-tolylthio analogue (16) was obtained by an alternative procedure involving ring-opening of 6,2′-O-cyclouridine (15).     

Fat production byTrichoderma reesei

Summary Snake flask experiments were carried out as a preliminary study of fat formation byTrichoderma reesei (formallyviride) QM 9123 using a glucose based medium. The maximum quantity of fat production was 16% of the dry weight. The pH of the medium, as much as its composition, appeared to influence the quantity of fat that was produced. The fatty acids formed by the hydrolysis of the fats were found to be mainly unsaturated, with the 182 (linoleic) acid predominating.     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

A self-referential model for the formation of the genetic code

A model for the formation of the genetic code is presented where protein synthesis is directed initially by tRNA dimers. Proteins that are resistant to degradation and efficient RNA-binders protect the RNAs. Replication becomes elongational producing poly-tRNAs from which the mRNAs and ribosomes are derived. Attributions are successively fixed to tRNAs paired through the perfect palindromic anticodons, with the same bases at the extremities (5′ANA: UNU 3′; GNG: CNC; principal dinucleotides, pDiN). The 5′ degeneracy is then developed. The first pairs to be encoded correspond to the hydropathy correlation outliers (Gly-CC: Pro-GG and Ser-GA: Ser-CU) and to the sector of homogeneous pDiN, composed by two pyrimidines or two purines. These amino acids are preferred in the N-ends of proteins, stabilizers of proteins against catabolism and strong RNA-binders. The next pairs complete the sector of homogeneous pDiN (Asp, Glu-UC: Leu-AG and Asn, Lys-UU: Phe-AA). This set of nine amino acids forms the protein cores with the predominant aperiodic conformation. Next enter the pairs with mixed pDiN (one purine and one pyrimidine), the RY attributions composing the protein N-ends and the YR attributions the C-ends. The last pair contains the main punctuation signs (Ile, Met, iMet-AU: Tyr, Stop-UA). The model indicates that genetic information emerged during the process of formation of the coding/decoding system and that genes were defined by the proteins. Stable proteins constructed the nucleoprotein system by binding to the RNAs that produced them. In this circular rationale, genes are memories in a metabolic system for production of proteins that stabilize it. The simplicity and the highly deterministic character of the process suggest that the Last Universal Common Ancestor populations could be composed, in early stages, of lineages bearing similar genetic codes.     

Chemical modification of lipase with ferromagnetic modifier — A ferromagnetic-modified lipase

Summary A ferromagnetic modifier was prepared by reacting ferrous(Fe²⁺)- and ferric(Fe³⁺)-ions with polyethylene glycol having two carboxyl groups (MW:2000) at pH 8.0–8.5. Lipase fromPseudomonas fragi 22–39B was coupled with the modifier using water-soluble carbodiimide. The modified lipase, which was dispersed into buffered solutions in the size range of 30–70 nm, exerted the hydrolytic activity of 8.0 U/mg. In a magnetic field of 250 Oe, the ferromagnetic-modified lipase was readily recovered from the colloidal solution.     

The number and nature of , -unsaturated amino acids in subtilin

In subtilin, a peptide produced by Bacillus subtilis, there are present three α,β-unsaturated amino acids, namely, two residues of dehydroalanine and one residue of β-methyldehydroalanine (dehydrobutyrine). Subtilin and nisin, a polypeptide produced by Streptococcus lactis, thus have in common not only the COOH-terminal sequence dehydroalanyllysine but also the number and nature of α,β-unsaturated amino acids.     

Esterification of chiral secondary alcohols with fatty acid in organic solvents by polyethylene glycol-modified lipase

Lipase from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase (PEG-lipase) was soluble and active in organic solvents such as benzene and 1,1,1-trichloroethane. PEG-lipase catalyzed esterification of chiral secondary alcohols with fatty acids in benzene and exhibited preference for R isomers over S isomers. Km and Vmax values for each isomer of various alcohols were obtained by kinetic study of the esterification in benzene. PEG-lipase-catalyzed esterification leads to optical resolution of a racemic alcohol.     

Purification and structure of the host-specific toxin from Helminthosporium carbonum race 1

The host-specific toxin from Helminthosporiumcarbonum race 1 was purified from culture filtrates by solvent extraction, gel filtration, and high pressure liquid chromatography. High resolution mass spectrometry of the purified toxin gave a MW of 436.2318 and an elemental composition C₂₁H₃₂N₄O₆. Amino acid analysis and proton and¹³C-NMR indicated a peptide containing four amino acids. Their sequence was determined by gas chromatography mass spectrometry. Finally, digestion of the amino acids with D- and L-amino acid oxidases gave the complete structure cyclo[(L-2-amino-9, 10-epoxy-8-oxodecanoyl)-D-prolyl-L-alanyl-L-alanyl].     

Bile acid catabolites accumulated by transposon-induced mutants ofPseudomonas putida: Production of hydroxylated 1,4-androstadiene-3,17-diones

Summary Transposon mutagenesis of a bile acid-utilizingPseudomonas putida strain generated 5 classes of mutants based on their ability to accumulate steroild catabolites. Bile acids (up to 5%) could be fermented using mutant strains, to the appropriate hydroxy-1,4-androstadiene-3,17-dione type product in yields close to theoretical.     

Butanol production from cellulosic substrates by sequential co-culture ofClostridium thermocellum andC.acetobutylicum

A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to fuels and chemicals. Growth ofClostridium acetobutylicum on solka floc (or a mixture of solka floc and aspenwood xylan), in co-culture withC.thermocellum, resulted in the efficient utilization of all the hydrolysis products derived from the lignocellulosic substrates. This co-culture approach resulted in a 1.7–2.6 fold increase in the total fermentation products formed. The majority of the fermentation products were acids and not solvents, however the solventogenesis step could be induced by the addition of butyric acid to the fermentation medium.