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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| Format: | Thesis |
| Language: | English English |
| Published: |
2003
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| Subjects: | |
| 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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| Summary: | 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. |
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