Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Formation of amino acids from aliphatic nitriles and aliphatic amines by contact glow discharge electrolysis

Contact glow discharge electrolyses (CGDE) were carried out relative to the prebiotic formation of amino acids by amination of aliphatic nitrile in aqueous ammoniacal solution, and by cyanization of aliphatic amine by sodium cyanide. The CGDE of propionitrile by amination followed by hydrolysis resulted in the formation of glycine, alanine and β-alanine. The reaction of ethylamine by cyanization, gave glycine, alanine, β-alanine, aspartic acid, and serine. In these reactions, a relatively high ratio of glycine was observed. This could be explained by the cleavage of the α,β-carbon bond, which was broken easily, due to the strong electron-attracting property of the nitrile group of propionitrile and the resulting α-aminopropionitrile.     

Conversion of aliphatic 2-acetoxynitriles by nitrile-hydrolysing bacteria

The enzymatic hydrolysis of the nitrile group of different 2-acetoxynitriles was investigated in order to obtain catalysts that chemoselectively hydrolyse nitriles in the presence of ester groups. The biotransformation of four 2-acetoxynitriles [2-acetoxybutenenitrile (ABN), 2-acetoxyheptanenitrile (AHN), 2-acetoxy-2-(2-furyl)acetonitrile (AFN), and 2-acetoxy-2,3,3-trimethylbutanenitrile (ATMB)] by different bacterial strains that synthesise nitrilases or nitrile hydratases was studied. ABN, AHN and AFN were converted by various microorganisms belonging to different bacterial genera (e.g. Pseudomonas or Rhodococcus) expressing either nitrilase or nitrile hydratase activities. In contrast, no metabolism of the sterically hindered substrate ATMB was observed. All wild-type strains investigated formed considerable amounts of cyanide and aldehydes from the 2-acetoxynitriles. This indicated the presence of esterases converting the 2-acetoxynitriles to 2-hydroxynitriles, which then spontaneously decomposed to the corresponding aldehydes and cyanide. In order to suppress unwanted side-reactions, biotransformations were performed with recombinant Escherichia coli strains that heterologously expressed nitrilase activities originating from Pseudomonas, Rhodococcus, or Synechocystis strains. The attempted conversion of the 2-acetoxynitriles to almost stoichiometric amounts of the corresponding 2-acetoxycarboxylic acids was finally achieved by using either a recombinant E. coli strain that highly overexpressed the nitrilase gene from the pseudomonad or the purified enzyme derived from this strain.     

Interaction of aliphatic amines with glycogen phosphorylase

A number of aliphatic amines was shown to stimulate AMP-dependent activity of phosphorylase b. The extent of stimulation depends on the molecular structure of amines. For linear amines, the longer the linear chain, the greater the stimulation observed. High concentrations of amines were able to induce a small activation of phosphorylase b in the absence of AMP. Kinetic studies of phosphorylase b indicated that the presence of n-hexylamine (a) results in lowering Km values for AMP and glucose 1-phosphate, (b) increases maximal velocity of the enzyme, and (c) modifies the glucose 6-phosphate, ATP, caffeine, and glucose binding sites of the enzyme by increasing the inhibition constants for these inhibitors. In contrast, the activity of phosphorylase b' is not altered by n-hexylamine. This fact suggests the possibility that amines interact with the N-terminal tail of phosphorylase b chain.     

Inclusion complex of beta-chitin and aliphatic amines

Inclusion complexation of beta-chitin with linear aliphatic amines was studied by X-ray diffraction. All tested amines, C3 to C8 monoamines and C2 to C7 diamines with terminal amino groups, reversibly formed crystalline complexes with beta-chitin by immersion of dry chitin in pure liquid. Complex formation caused linear increase in the 010 sheet spacing of beta-chitin depending on the carbon number of amine. The complexes could be classified as type I and type II according to the increment of sheet spacing against carbon number. All monoamines formed type II complexes. In dry conditions, diamine formed a type I complex though the type of diamine complex differed for guest species in wet conditions. Based on the unit cell dimension and thermogravimetry, type II and type I are likely to correspond to guest-host (amine-chitobiose) ratios of 2:1 and 1:1, respectively. These differences seem to arise from varied interactions between functional groups of chitin and amines.     

Determination of trimethylamine and related aliphatic amines in human urine by head-space gas chromatography

A rapid and simple assay procedure employing head-space gas chromatography has been developed for the routine quantification of volatile methylamines and stable trimethylamine N-oxide present in human urine. This assay will enable the rapid screening of patients and aid the diagnosis of fish odour syndrome.     

Spectrophotometric method for estimation of aliphatic primary amines in biological samples

A method based on Rimini test for aliphatic amines was studied and developed for quantitative estimation of aliphatic primary amines. The method involves action of the amine with acetone to form schiff base which complexes with sodium nitroprusside to give violet colour. The absorption maximum in the visible range of the spectrum, for the reaction mixture was found to be 550 nm. The pH (8–11) and reaction time scan for the assay were optimized. A linear relation of concentration (0.2–3 mg/mL) of amine against absorbance at 550 nm was established. Interference due to other reaction components was negligible (±0.02 mg/mL) as compared to the sample in buffer. 1, 3-dimethyl butylamine was used as the model amine and the method was applied to other amines; it was observed that when electron-withdrawing substituents are present in the molecule the reaction is retarded, as the incubation time was longer. This method is useful for estimation of aliphatic primary amine in biological samples.     

Kinetic effect of some aliphatic amines on yeast alcohol dehydrogenase.

Initial rate studies of ethanol oxidation catalyzed by yeast alcohol dehydrogenase (EC 1.1.1.1) were carried out in the presence of varying concentrations of aliphatic amines over the pH range from 8.0 to 10.5. Aliphatic amines either activate or inhibit the enzyme depending on whether the pH is greater or less than 9.5 suggesting that the protonated amines activate and the nonprotonated amines inhibit the enzyme. Aliphatic amines activate yeast alcohol dehydrogenase by decreasing Kb while they inhibit the enzyme by increasing both Ka and Kia. When both protonated and nonprotonated amines are present in solution, either overall activation or inhibition will be observed depending on the relative concentration of the two amine species.