N Acetyl L Cysteine - The Conspriracy > 자유게시판

The amino acid acetyl-l-carnitine, or ALCAR, outcomes from the synthesis of carnitine, while the amino acid cysteine leads to NAC. The radius of the feeding auriole around the take a look at colony (halo) was taken to be a semiquantitative measure of the cysteine secretion by the check pressure. The zone of growth of the indicator pressure round the actual inoculation streak (halo) serves as a semiquantitative measure of the cysteine secretion. For the purpose of expressing the altered serine acetyltransferase enzyme, the feedback-resistant cys-E alleles are reworked into a number pressure using customary strategies. FIG. 5 shows the amino acid sequence of E. coli serine acetyltransferase. By the use of using the previously mentioned mutagenesis methods, a number of nucleotides within the DNA sequence are modified such that the amino acid sequence which is now encoded by the gene exhibits no less than one mutation in the sequence region from place ninety seven up to and together with the amino acid in place 273, or a minimum of one deletion is present within the carboxyterminal sequence region beginning with the amino acid in position 227, where position 1 is the initiating methionine in FIG. 5 (SEQ ID NO: 1) and the place the mutation of Met to Ile in position 256 is excluded.

Preferably, the gene of the native, cysteine-delicate serine acetyltransferase is inactivated in the host strain, thereby ensuring that it is only the cysteine-insensitive serine acetyltransferase, which has been introduced into the particular strain by transformation, which is synthesized. For the dedication, use was manufactured from S30 extracts (cell homogenates centrifuged at 30,000 g and 4° C. for 20 minutes) of the selected revertants, the starting strains and the comparability pressure, E. coli W3110 (ATTC 27325). Quite a few revertants were discovered whose serine acetyltransferase exercise nonetheless exhibited significant residual activity (Ki worth of between 5 and 50 μM) within the presence of differing concentrations of the inhibitor, L-cysteine. Mutants possessing the specified properties (insensitivity of the serine acetyltransferase to cysteine) are sought among the revertants of cysteine-auxotrophic cysE E. coli strains. Preferably, the protein sequence of a novel serine acetyltransferase incorporates the amino acid substitution of at least one of the cysE mutants specified in Tab. FIG. 10 shows a nucleotide sequence discussed on page 21 of the specification. Determination of the enzymic exercise and the inhibitor fixed, Ki, and likewise the cross-feeding check, had been carried out in analogy with the outline in Example 1. DNA sequence evaluation was used to affirm that the deletions had been correctly launched.

It has been found, surprisingly, that the novel amino acid substitutions and/or amino acid deletions of the carboxyl terminus of the serine acetyltransferase result in a diminution in the cysteine sensitivity whereas at the same time allowing adequate enzymic activity to be retained. An enzyme exercise check is carried out on the selected strains in order to find out the extent of the cysteine tolerance of the altered serine acetyltransferase. Such strains of microorganisms are characterized by the fact that they possess a cysteine metabolism which is deregulated by at the least one suggestions-resistant cysE allele. So as to determine the extent of the cysteine insensitivity of the altered enzyme, the secretion of cysteine by the pressure is to begin with measured in a semiquantitative, so-referred to as cross-feeding test. Bacteria are ideally appropriate for preparing a novel strain. L-cysteine-associated products are all products which are derived from L-cysteine, i.e. sulfur-containing compounds which require L-cysteine for his or her preparation. The suggestions-resistant cys-E alleles render it possible to abolish the management at an vital biosynthetic control level, thereby amplifying the manufacturing of numerous compounds which are situated downstream of this control level. So as to find out the power to secrete cysteine in a liquid medium by quantitatively determining cysteine, 50 chosen cysE revertants were incubated in 20 ml of standard production medium at 30° C. and at 170 rpm for a period of forty eight hours.

The standard manufacturing medium consisted of 15 g/L glucose, 0.08 g/L bactotryptone, 0.04 g/L yeast extract, 5 mg/L vitamin B1, 3 g/L KH2 PO4, 12 g/L K2 HPO4, 0.3 g/L MgSO4×7 H2 O, 0.1 g/L NaCl, 5 g/L (NH4)2 SO4, 14.7 mg/L CaCl2×2 H2 O, 2 mg/L FeSO4×2 H2 O, 1 g/L Na3 citrate×2 H2 O, 5 g/L Na2 S2 O3×5 H2 O and 1 ml/L hint factor answer (cf. Subsequent to the mutagenesis, which has been carried out, for instance, as described, the mutants having the desired phenotype are chosen, for instance via plating on to cysteine-free medium and subsequently figuring out the extent to which the mutated serine acetyltransferase is sensitive to cysteine. The above-described enzyme take a look at is appropriate for determining the cysteine sensitivity of any serine acetyltransferase enzyme, together with the enzymes which have a modified carboxyl terminus. A sample (10 μl) was in every case eliminated after 24 and 48 hours and diluted, where applicable, N-Acetyl-L-Cysteine 98% manufacturers and the cysteine focus within the cell-free supernatant was determined calorimetrically using the strategy of Gaitonde, M. K. (1967), Biochem.