Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Pae, Ki Moon; Ryu, Ok Hee; Yoon, Hyun Sook; Shin, Changsoo

doi:10.1007/bf01027018

Cited by 14 publications

(6 citation statements)

References 4 publications

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“…Pae et al (1992) reported a CD gene mutant strain by ultraviolet mutagenesis, and its L-cysteine productivity was increased from 67% to 95%. It also has been demonstrated that disruption of CD genes was significantly effective at achieving L-cysteine overproduction in E. coli ((Awano et al, 2003;Awano et al, 2005), so we are presently continuing this research in order to construct a CD-gene-disruption strain to increase the L-cysteine production.…”

Section: Discussionmentioning

confidence: 98%

Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138

Bai

Yang

et al. 2007

Front. Biol. China

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L-cysteine desulfhydrase (CD) plays an important role in L-cysteine decomposition. To identify the CD gene in Pseudomonas sp. TS1138 and investigate its effect on the L-cysteine biosynthetic pathway, the CD gene was cloned from Pseudomonas sp. TS1138 by polymerase chain reaction (PCR) method. The nucleotide sequence of CD gene was determined to be 1,215 bp, and its homology with other sequences encoding CD was analyzed. Then the CD gene was subcloned into pET-21a(+) vector and expressed in Escherichia coli (E. coli) by isopropyl-b-D-thiogalactopyranoside (IPTG) inducement. The recombinant CD was purified by Ni-NTA His-Bind resin, and its activity was identified by the CD activity staining. The enzymatic properties of the recombinant CD were characterized and its critical role involved in the L-cysteine biosynthetic pathway was also discussed.

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Section: Discussionmentioning

confidence: 98%

Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138

Bai

Yang

et al. 2007

Front. Biol. China

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“…The pH was adjusted to 7.0 with 6 N NaOH. Seed cultures and main cultures were carried out as previously described in [5,8].…”

Section: Medium and Cultivationmentioning

confidence: 99%

“…The L-cysteine content was measured by the GAITONDE method [Ill. The amounts of D,L-ATC and L-cystine were measured using a HPLC (GILSON) [8]. The content of Lcystine was expressed as an L-cysteine equivalent, where one mole of L-cystine corresponds to two moles of L-cysteine.…”

Section: Measurement Of D L-atc L-cysteine and L-cystine Contentmentioning

confidence: 99%

“…Thus, since the conversion of L-ATC to L-cysteine can be accomplished using L-ATC hydrolase as well as hydroxylamine, and the conversion of D-ATC to L-ATC is reversible and fast, the overall reaction rate of D-ATC to L-cysteine is considered to be controlled by the conversion step of L-ATC to S-carbamyl-L-cysteine. Both final products, L-cysteine and L-cystine, inhibit the conversion of L-ATC to S-carbamyl-Lcysteine using L-ATC hydrolase [8]. LCysteine is easily converted to L-cystine in air, but L-cysteine remains largely unconverted in the anoxic conditions of a nitrogen atmosphere.…”

Section: Introductionmentioning

confidence: 99%

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Effects of anoxic conditions on the enzymatic conversion of D,L‐2‐amino‐thiazoline‐4‐carboxylic acid to L‐cystine

Nam

Ryu

Park

et al. 1997

Acta Biotechnologica

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The effects of anoxic conditions on product inhibition and the stability of L-ATC hydrolase were investigated in the conversion of D,L-2-amino-A2-thiamline-4-carboxylic acid (D, L-ATC ) to L-cystine using the cell free extract enzyme of Pseudomonas sp. in the presence of hydroxylamine. At L-cysteine equivalent levels, where one mole of L-systine was counted as two moles of L-cysteine, L-cystine inhibited the L-ATC hydrolase reaction to a greater extent than Lcysteine. In air. the product occurred predominantly as L-cystine (94.9%), whereas in a nitrogen atmosphere the product occurred as a mixture of L-cysteine (39.3%) and L-cystine (40.7%). As a result, less product inhibition took place in nitrogen. The activity of L-ATC hydrolase was almost fully lost after 20 h of incubation by shaking at 30 "C in air, but considerable activity remained under the anoxic conditions of nitrogen. A kinetic analysis of the reactions confirmed that reduced product inhibition and enhanced enzyme stability in nitrogen result in a more efficient enzyme reaction. The inactivation rate constant (kl) was estimated to be 0.1 1 h -1 in nitrogen and 0.22 -1 in air, indicating that the stability of L-ATC hydrolase in nitrogen was greater than in air. The values of the Kp and KPZ constants related to product inhibition were 43.36 mM and 30.48 mM for L-cysteine and 2-cystine, respectively, where higher values were an indication of less product inhibition. The value of the rate constant (kz) for the oxidation of L-cysteine to L-cystine was 0.09 h -in nitrogen and 1.01 h -in air, suggesting that the oxidation of L-cysteine to L-cystine proceeds faster in air than in nitrogen.

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“…Biotechnological production processes have been used for industrial production of amino acids for about 50 years now (1,3,4,5,9,10,12,14,17,19,20,21,24,27,28,32,33,34,35,42,43,44,52,58).…”

Section: Production Of Amino Acids By Biotechnology Methodsmentioning

confidence: 99%

Biotechnology in the Production of Pharmaceutical Industry Ingredients: Amino Acids

Ivanov

Stoimenova

Obreshkova

et al. 2013

Biotechnology & Biotechnological Equipment

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Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Cited by 14 publications

References 4 publications

Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138

Cloning, expression and characterization of L-cysteine desulfhydrase gene from Pseudomonas sp. TS1138

Effects of anoxic conditions on the enzymatic conversion of D,L‐2‐amino‐thiazoline‐4‐carboxylic acid to L‐cystine

Biotechnology in the Production of Pharmaceutical Industry Ingredients: Amino Acids

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