One-step elimination of l-cysteine desulfhydrase from crude enzyme extracts of Pseudomonas sp. TS1138 using an immunomagnetic affinity matrix improves the enzymatic production of l-cysteine

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 853; no. 1-2; pp. 247 - 253
• Main Authors: Yu, Yangsheng, Bai, Gang, Liu, Chunqin, Cao, Yu, Geng, Peng, Yang, Wenbo
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.06.2007; Elsevier Science
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biological and medical sciences; Cystathionine gamma-Lyase - genetics; Cystathionine gamma-Lyase - isolation & purification; Cystathionine gamma-Lyase - metabolism; Cysteine - metabolism; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; General pharmacology; Immunomagnetic separation; l-Cysteine; l-Cysteine desulfhydrase; Magnetic cellulose microspheres; Magnetics; Medical sciences; Microscopy, Electron, Scanning; Microspheres; Pharmacology. Drug treatments; Pseudomonas - enzymology; Pseudomonas - genetics; Pseudomonas sp. TS1138; Pyruvates - metabolism; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; l-Cysteine; Immunomagnetic separation; l-Cysteine desulfhydrase; Magnetic cellulose microspheres; Pseudomonas sp. TS1138; Pseudomonadales; Cystathionine γ-lyase; Thiol; Cysteine; Immunomagnetic method; Microsphere; Enzyme; Elimination; Lyases; Extract; Pseudomonas; Sulfur containing aminoacid; L-Cysteine; Cellulose derivatives; Carbon-sulfur lyases; Bacteria; Microparticle; Pseudomonadaceae; Affinity; Matrix system; L-Cysteine desulfhydrase
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2007.03.021

In this study, a high efficiency immunomagnetic affinity matrix was developed to eliminate l-cysteine desulfhydrase (CD), which decomposes l-cysteine, in crude enzyme extracts from Pseudomonas sp. TS1138. After cloning and expression in Escherichia coli, recombinant CD was purified to raise polyclonal antibodies from mice. The anti-CD antibody was cross-linked to staphylococcal protein A-magnetic cellulose microspheres (MCMS) with dimethyl pimelimidate (DMP). The natural CD was eliminated from the crude enzyme extracts by treatment with the cross-linked antibody-protein A-MCMS, resulting in a high level of l-cysteine production. The conversion rate of dl-2-amino-Δ2-thiazoline-4-carboxylic acid (dl-ATC) to l-cysteine increased significantly from 61.9 to 96.2%. The cross-linked antibody-protein A-MCMS showed its durability after repetitive use, maintaining a constant binding capacity for CD during five cycles. This study may lead to a convenient and cost-efficient method to produce l-cysteine by enzymatic conversions.