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Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)

L-Methionine y-Iyase (EC 4.4.1.11; LMGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of L-methionine to aketobutyrate, methanethiol and ammonia by an a,r-elimination reaction. Seventy nine LMGL-producing microorganisms isolates were screened from s...

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Main Author: Lim, Leng Choo
Format: Thesis
Language:English
English
Published: 2003
Subjects:
Methionine.
Vitamin B6.
Online Access:http://psasir.upm.edu.my/id/eprint/8508/1/FSMB_2003_13_A%20D.pdf
http://psasir.upm.edu.my/id/eprint/8508/
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spelling my.upm.eprints.85082022-01-26T01:15:08Z http://psasir.upm.edu.my/id/eprint/8508/ Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25) Lim, Leng Choo L-Methionine y-Iyase (EC 4.4.1.11; LMGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of L-methionine to aketobutyrate, methanethiol and ammonia by an a,r-elimination reaction. Seventy nine LMGL-producing microorganisms isolates were screened from six local sources by the 5'5-dithiobis (2-nitrobenzoic acid) (DTNB) test. The six local sources were soil samples from around the Faculty of Food Science and Biotechnology and Central Research Laboratory, Universiti Putra Malaysia and Kuantan sea coast, soil and water samples from hot springs in Ulu Legong, Baling, Kedah and Pedas, Negeri Sembilan and intestine samples from chicken. A simple and convenient colorimetric screening method, the DTNB test detects methanethiol, which reduces DTNB contained in an agar-plate medium to form yellow colour aryl mercaptan (4 thiol-2-nitro-benzoate) around the colony of a bacterium that is able to produce LMGL. LMGL was detected from 45 (57%) of the bacterial isolates by 3-methyl-2-benzothiazolone hydrazone (MBTH) assay. LMGL activity was quantitatively assayed by determining the amount of a-ketobutyrate produced spectrophotometricalIy at 320 run after derivatization with MBTH. Twelve relatively high producers of LMGL were identified by Gram stain, 10 types of biochemical tests consisting of potassium hydroxide (KOH), catalase, oxidase, indole, citrate utilization, phenylalanine deaminase and urease tests and triple sugar iron agar (ISIA), nutrient agar and MacConkey agar reactions, and by using the Biolog test kits (Biolog, Inc., Hayward, Calif.). Enterobacter nimipressuralis, Enterobacter intermedius, Pseudomonas pyrrocinia, Ratstonia pickettii and Citrobacter freundii (C freundii) were found to be new sources for LMGL while the remaining two were Escherichia coli and Bacillus cereuslthuringiensis. The methionine-utilizing enzyme was partially purified from C freundii (KP25) isolated from soil samples of Kuantan sea coast, which contained the highest activity. The purification scheme, involving dialysis, removal of nucleic acid with deoxyribonuclease I (DNase I) and ammonium sulfate [(NH₄)₂SO₄] precipitation resulted in a purification fold of 0.6 with a recovery of 22.6% and a specific activity of 0.02 U/mg, all using methionine as the substrate. It was found that the partially purified enzyme extract from C freundii (KP25) catalyzed D-amino acids better than L-amino acids and also degraded cysteine and its S-substituted derivatives such as more effectively than methionine and its S-substituted derivatives. Hence, the result on substrate specificity of the lyase present in the enzyme extract shows the probable presence of D-cysteine desulfbydrase (EC 4.4.1.15) and the absence of LMGL. Crude enzyme extract from C. freundii (KP25) was characterized by using D- and L-cysteine instead of DL-methionine as the substrates. The temperature and pH optimum of the crude enzyme extract were 45°C and pH 9.0 in 125 mM glycine-sodium hydroxide (NaOH) buffer with each D- and L-cysteine as the substrate. 2003 Thesis NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/8508/1/FSMB_2003_13_A%20D.pdf Lim, Leng Choo (2003) Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25). Masters thesis, Universiti Putra Malaysia. Methionine. Vitamin B6. English
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
English
topic Methionine.
Vitamin B6.
spellingShingle Methionine.
Vitamin B6.
Lim, Leng Choo
Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)
description L-Methionine y-Iyase (EC 4.4.1.11; LMGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of L-methionine to aketobutyrate, methanethiol and ammonia by an a,r-elimination reaction. Seventy nine LMGL-producing microorganisms isolates were screened from six local sources by the 5'5-dithiobis (2-nitrobenzoic acid) (DTNB) test. The six local sources were soil samples from around the Faculty of Food Science and Biotechnology and Central Research Laboratory, Universiti Putra Malaysia and Kuantan sea coast, soil and water samples from hot springs in Ulu Legong, Baling, Kedah and Pedas, Negeri Sembilan and intestine samples from chicken. A simple and convenient colorimetric screening method, the DTNB test detects methanethiol, which reduces DTNB contained in an agar-plate medium to form yellow colour aryl mercaptan (4 thiol-2-nitro-benzoate) around the colony of a bacterium that is able to produce LMGL. LMGL was detected from 45 (57%) of the bacterial isolates by 3-methyl-2-benzothiazolone hydrazone (MBTH) assay. LMGL activity was quantitatively assayed by determining the amount of a-ketobutyrate produced spectrophotometricalIy at 320 run after derivatization with MBTH. Twelve relatively high producers of LMGL were identified by Gram stain, 10 types of biochemical tests consisting of potassium hydroxide (KOH), catalase, oxidase, indole, citrate utilization, phenylalanine deaminase and urease tests and triple sugar iron agar (ISIA), nutrient agar and MacConkey agar reactions, and by using the Biolog test kits (Biolog, Inc., Hayward, Calif.). Enterobacter nimipressuralis, Enterobacter intermedius, Pseudomonas pyrrocinia, Ratstonia pickettii and Citrobacter freundii (C freundii) were found to be new sources for LMGL while the remaining two were Escherichia coli and Bacillus cereuslthuringiensis. The methionine-utilizing enzyme was partially purified from C freundii (KP25) isolated from soil samples of Kuantan sea coast, which contained the highest activity. The purification scheme, involving dialysis, removal of nucleic acid with deoxyribonuclease I (DNase I) and ammonium sulfate [(NH₄)₂SO₄] precipitation resulted in a purification fold of 0.6 with a recovery of 22.6% and a specific activity of 0.02 U/mg, all using methionine as the substrate. It was found that the partially purified enzyme extract from C freundii (KP25) catalyzed D-amino acids better than L-amino acids and also degraded cysteine and its S-substituted derivatives such as more effectively than methionine and its S-substituted derivatives. Hence, the result on substrate specificity of the lyase present in the enzyme extract shows the probable presence of D-cysteine desulfbydrase (EC 4.4.1.15) and the absence of LMGL. Crude enzyme extract from C. freundii (KP25) was characterized by using D- and L-cysteine instead of DL-methionine as the substrates. The temperature and pH optimum of the crude enzyme extract were 45°C and pH 9.0 in 125 mM glycine-sodium hydroxide (NaOH) buffer with each D- and L-cysteine as the substrate.
format Thesis
author Lim, Leng Choo
author_facet Lim, Leng Choo
author_sort Lim, Leng Choo
title Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)
title_short Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)
title_full Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)
title_fullStr Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)
title_full_unstemmed Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25)
title_sort characterization of a sulfuramino acid lyase from citrobacter freundii (kp25)
publishDate 2003
url http://psasir.upm.edu.my/id/eprint/8508/1/FSMB_2003_13_A%20D.pdf
http://psasir.upm.edu.my/id/eprint/8508/
_version_ 1724075529577955328
score 13.211869

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