N Acetyl L Cysteine - The Conspriracy > 플랫폼 수정 및 개선 진행사항

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The amino acid acetyl-l-carnitine, or ALCAR, results 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 take a look at pressure. The zone of development of the indicator N-Acetyl-L-Cysteine 98% USA pressure round the particular inoculation streak (halo) serves as a semiquantitative measure of the cysteine secretion. For the purpose of expressing the altered serine acetyltransferase enzyme, the suggestions-resistant cys-E alleles are reworked into a number strain utilizing customary strategies. FIG. 5 reveals the amino acid sequence of E. coli serine acetyltransferase. Via utilizing the previously mentioned mutagenesis strategies, a number of nucleotides in the DNA sequence are modified such that the amino acid sequence which is now encoded by the gene exhibits at least one mutation in the sequence region from position ninety seven as much as and together with the amino acid in position 273, or at the least one deletion is present in the carboxyterminal sequence region starting with the amino acid in place 227, the place 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 just the cysteine-insensitive serine acetyltransferase, which has been introduced into the particular pressure by transformation, which is synthesized. For the dedication, use was made 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). Various revertants were discovered whose serine acetyltransferase exercise nonetheless exhibited significant residual activity (Ki value 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 many revertants of cysteine-auxotrophic cysE E. coli strains. Preferably, the protein sequence of a novel serine acetyltransferase accommodates the amino acid substitution of a minimum of one of the cysE mutants specified in Tab. FIG. 10 shows a nucleotide sequence discussed on web page 21 of the specification. Determination of the enzymic exercise and the inhibitor fixed, Ki, and in addition the cross-feeding check, have been carried out in analogy with the outline in Example 1. DNA sequence evaluation was used to affirm that the deletions had been appropriately introduced.

It has been discovered, surprisingly, that the novel amino acid substitutions and/or amino acid deletions of the carboxyl terminus of the serine acetyltransferase lead to a diminution within the cysteine sensitivity whereas at the identical time allowing adequate enzymic activity to be retained. An enzyme exercise check is carried out on the chosen strains so as to determine the extent of the cysteine tolerance of the altered serine acetyltransferase. Such strains of microorganisms are characterized by the truth that they possess a cysteine metabolism which is deregulated by at the very 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-called cross-feeding take a look at. Bacteria are ideally appropriate for getting ready a novel strain. L-cysteine-related merchandise are all products which are derived from L-cysteine, i.e. sulfur-containing compounds which require L-cysteine for their preparation. The suggestions-resistant cys-E alleles render it potential to abolish the management at an important biosynthetic management level, thereby amplifying the manufacturing of numerous compounds that are situated downstream of this control level. So as to find out the flexibility to secrete cysteine in a liquid medium by quantitatively figuring out cysteine, 50 chosen cysE revertants had been incubated in 20 ml of commonplace manufacturing medium at 30° C. and at 170 rpm for a interval of forty eight hours.

The standard production medium consisted of 15 g/L glucose, 0.08 g/L bactotryptone, 0.04 g/L yeast extract, 5 mg/L vitamin B1, three g/L KH2 PO4, 12 g/L K2 HPO4, 0.Three 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 solution (cf. Subsequent to the mutagenesis, which has been carried out, for example, as described, the mutants having the desired phenotype are chosen, for example by means of plating on to cysteine-free medium and subsequently figuring out the extent to which the mutated serine acetyltransferase is delicate to cysteine. The above-described enzyme test is appropriate for figuring out the cysteine sensitivity of any serine acetyltransferase enzyme, including the enzymes which have a modified carboxyl terminus. A sample (10 μl) was in every case eliminated after 24 and forty eight hours and diluted, where applicable, and the cysteine focus in the cell-free supernatant was determined calorimetrically utilizing the tactic of Gaitonde, M. K. (1967), Biochem.