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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my-upm-ir.85082022-01-26T01:15:08Z Characterization of a Sulfuramino Acid Lyase from Citrobacter Freundii (KP25) 2003 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. Methionine. Vitamin B6. 2003 Thesis http://psasir.upm.edu.my/id/eprint/8508/ http://psasir.upm.edu.my/id/eprint/8508/1/FSMB_2003_13_A%20D.pdf text en public masters Universiti Putra Malaysia Methionine. Vitamin B6. Food Science and Technology Mohd. Ghazali, Hasanah English |
| institution |
Universiti Putra Malaysia |
| collection |
PSAS Institutional Repository |
| language |
English English |
| advisor |
Mohd. Ghazali, Hasanah |
| 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 |
| qualification_level |
Master's degree |
| 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) |
| granting_institution |
Universiti Putra Malaysia |
| granting_department |
Food Science and Technology |
| publishDate |
2003 |
| url |
http://psasir.upm.edu.my/id/eprint/8508/1/FSMB_2003_13_A%20D.pdf |
| _version_ |
1747810821504761856 |