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Purification and Characterization

▪ Abstract In procaryotes, l-carnitine may be used as both a carbon and nitrogen source for aerobic growth, or the carbon chain may be used selectively following cleavage of trimethylamine. Under anaerobic conditions and in the absence of preferred substrates, some bacteria use carnitine, via crotonobetaine, as an electron acceptor. Formation of trimethylamine and γ-butyrobetaine (from reduction of crotonobetaine) from l-carnitine by enteric bacteria has been demonstrated in rats and humans. Carnitine is not degraded by enzymes of eukaryotic origin. In higher organisms, carnitine has specific functions in intermediary metabolism. Concentrations of carnitine and its esters in cells of eukaryotes are rigorously maintained to provide optimal function. Carnitine homeostasis in mammals is preserved by a modest rate of endogenous synthesis, absorption from dietary sources, efficient reabsorption, and mechanisms present in most tissues that establish and maintain substantial concentration gradients between intra...

Carnitine metabolism and its regulation in microorganisms and mammals

Chitin is the second most abundant organic and renewable source in nature, after cellulose. Chitinases are chitin-degrading enzymes. Chitinases have important biophysiological functions and immense potential applications. In recent years, researches on fungal chitinases have made fast progress, especially in molecular levels. Therefore, the present review will focus on recent advances of fungal chitinases, containing their nomenclature and assays, purification and characterization, molecular cloning and expression, family and structure, regulation, and function and application.

Review of fungal chitinases.

Environmental properties and effects of nonionic surfactant adjuvants in pesticides: a review

The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.

A novel endo-beta -1,3-glucanase, bgn13.1, involved in the mycoparasitism of trichoderma harzianum

An enzyme from Trichoderma harzianum dissolved the cell walls of a wide range of filamentous fungi belonging to Basidiomycotina, Ascomycotina, Deuteromycotina, and Zygomycotina and so could be used to make protoplasts. A lyophilized preparation of the Trichoderma enzyme had about 0.3 units/mg β-1,3-glucanase activity and 0.36 units/mg chitinase activity. About twice as many protoplasts were produced from different species of fungi by a single treatment with this enzyme than with combined commercial enzymes. The greatest number of protoplasts could be produced from most of the fungi by incubation for about 2 h t 30°C, but the number was decreased by incubation for more than 4 h or by use of a higher dose of the enzyme. An enzyme prepared by bentonite treatment from the original Trichoderma enzyme had less proteinase activity and protoplasts were fairly stable with this product during incubation for 8 h. Protoplasts produced by the proteinase-reduced preparation of the Trichoderma enzyme from three fungi regenerated at about 1.8 times the rate of those produced by the original enzyme.

A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum

Purification and some properties of an exo-β-1,3-glucanase from Trichoderma harzianum

clarify the properties of carnitine dehydrogenasc, In this report, we described the purification and some prepertics of the carnitine dehydregenase ot' X. transtucens. X. translucens (IFO 13558) was grown in a medium containing 20,Og of DL-carnitine chloride, 1.0g of K,HPO,, 1,Og of KH,Pe,, O.5g of MgSO,・7H,O, and e.5g of yeast extract in 1000ml of distilled water at pH 7.0. The cu}tivation was done at 30\"C for 20hr with reciprocal shaking. The harvested cells wcre suspended in 50mM Tris-HCI bufler (pH 7.S) and disrupted with an ultrasonie oscillator, Thc ccll-free extract was addeci te ammonium sulfate to 40% saturation and the precipitate was discarded. The precipitate formed by th¢ further addition of ammonium sulfate to 6e% saturation was collected and dissolved in 50mM Tris-HCI bufflrr (pH 7.5). The・dialyzed enzyme solution xrp'as put on a DEAE-cellulose column (4,0 × 55cm) equilibrated with 50mM Tris-HCI buffer (pH 7.S). The enzymc was eluted with a linear gradient of KCI CO -・O,5M)

Purification and Some Properties of Carnitine Dehydrogenase from Xanthomonas translucens

The degradation of choline by Candida tropicalis cells grown in a medium containing choline as a nitrogen source was examined. The degradation of choline by resting cells was stimulated by the addition of Cu2+ or glutathione, and inhibited by 2-mercaptoethanol or potassium cyanide. With feeding of [1,2-14C]choline in the resting cell reaction, the release of 14C-labelled ethylene glycol was observed on radio-gas-liquid chromatography. Ethylene glycol, as one of the degradation products, was also observed on thin-layer and gas-liquid chromatographies, and mass spectrometry. Thus, it is suggested that choline is converted to ethylene glycol and trimethylamine by C. tropicalis.

Yoshio Ichikawa

Papers

A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum

Purification and some properties of an exo-β-1,3-glucanase from Trichoderma harzianum

Purification and Some Properties of Carnitine Dehydrogenase from Xanthomonas translucens

Formation of ethylene glycol and trimethylamine from choline by Candida tropicalis

Degradation of Polyethylene Glycol Dodecyl Ethers by a Pseudomonad Isolated from Activated Sludge