Continuous l-cysteine production using immobilized cell reactors and product extractors

We investigated the conversion of D,L-2-amino-Δ (2)-thiazoline-4-carboxylic acid (D,L-ATC) to L-cysteine with Pseudomonas sp. ON-4a, an ATC-assimilating bacterium. Cysteine and N-carbamoylcysteine (NCC), but not S-carbamoylcysteine (SCC), were produced from D,L-ATC by a cell-free extract from the strain. These products were isolated from the reaction mixture and then identified as the L-form. Similar results were obtained with P. putida AJ3865 and unidentified strain TG-3, an ATC-assimilating bacteria. It became clear that L-NCC is an intermediate in the conversion of D,L-ATC to L-cysteine in these Pseudomonas strains. Furthermore, it was suggested that these bacteria have L-ATC hydrolase and L-NCC amidohydrolase.

https://www.tandfonline.com/doi/pdf/10.1271/bbb.62.2226

N-Carbamoyl-L-Cysteine as an Intermediate in the Bioconversion from D,L-2-Amino-Δ (2)-Thiazoline-4-Carboxylic Acid to L-Cysteine by Pseudomonas sp. ON-4a.

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.

Identification, Cloning, and Sequencing of the Genes Involved in the Conversion of D , L -2-Amino-Δ2-Thiazoline-4-Carboxylic Acid to …

N-carbamoyl-l-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50°C, respectively. The enzyme required Mn2+ ion for activity expression and was inhibited by EDTA, Hg2+ and sulfhydryl reagents. The enzyme was strictly specific for the l-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-l-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel l-carbamoylase, different from the known l-carbamoylases.

A novel N-carbamoyl-l-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-l-cysteine amidohydrolase expressed in Escherichia coli

A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.

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

The reaction steps involved in the bioconversion of a chemically synthesized precursor, acid (D,L-ATC), to L-cysteine and the properties of the involved enzymes were investigated. It was found that the conversion consisted of two steps, i. e., D,L-ATC to S-carbamyl-L-cysteine (S-C-L-cysteine) and S-C-L-cysteine to L-cysteine, and the S-C-L-cysteine was an intermediate between them. While the enzymes involved in the reactions were induced by the addition of D,L-ATC as an inducer, S-C-L-cysteine induced only the enzyme involved in the latter step. The conversion of S-C-L-cysteine to L-cysteine could be also carried out in the presence of hydroxylamine and its rate was much faster than that by the corresponding enzyme. On the other hand, L-cysteine (or L-cystine) was decomposed to evolve by the enzyme considered to be a kind of desulfhydrase. However, hydroxylamine was a perfect inhibitor for this enzyme.

Analysis of the Reaction Steps in the Bioconversion of D,L-ATC to L-Cysteine

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Ok-Hee Ryu

Author Tools

Chat about Author

Papers

Analysis of the Reaction Steps in the Bioconversion of D,L-ATC to L-Cysteine