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D-Carnitine Dehydrogenase dari
Agrobacteriumsp. 525a
Siswa SetyahadiPusat Teknologi BioindustriDeputi Teknologi Agroindustri dan Bioteknologi,
Badan Pengkajian dan Penerapan Teknologi
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http://www.sportindo.com/products/l-carnitine -
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L-Carnitine
L-Carnitine adalah sebuah molekul seperti vitamin yangmempunyai beberapa peranan penting dalam tubuh. L-
Carnitine adalah asam alpha-hydroxy yang utamanyaterletak di jaringan otot.
(CH3)3N+CH2CHCH2COO
-
OH (CH3)3N+CH2CHCH2COO
-
OH
L- Carnitine D- Carnitine
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FungsiFungsi utama dari carnitine di dalam tubuhadalah untuk mengatur oksidasi lemak
(membakar lemak). L-Carnitine bertanggungjawab untuk mengangkut lemak ke tungkupembakaran lemak dalam sel tubuh, yang
disebut dengan mitokondria. Lemaktidak akanbisa dioksidasi jika lemak tidak dapat mencapaimitokondria, sekeras apapun Anda berolahragaataupun melakukan diet.
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Produksi L-Carnitine dalam Tubuh
Tubuh bisa memproduksi sejumlah kecil L-Carnitine.Untuk memproduksi L-Carnitine, diperlukan asam amino
lisin dan metionin, serta beberapa vitamin seperti niasin,B6, dan vitamin C.Kekurangan salah satu nutrisi tersebut dapat menurunkanlevel L-Carnitine dalam tubuh dan menurunkan tingkatpembakaran lemak.
Lisin telah terbukti efektif untuk meningkatkan kadar L-Carnitine. Lisin merupakan prekursor asam amino yangmembentuk molekul carnitine.Diet vegetarian yang ketat membuat tubuh hanya
mendapatkan sedikit asupan L-Carnitine atau bahkan tidaksama sekali.
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Sumber Carnitine dalam Makanan
L-Carnitine ditemukan paling banyak di daging
dan makanan hewani. Daging merah adalah sumber carnitine
terbaik. Ayam dan kalkun juga mengandung
carnitine. Susu dan produk olahannya mengandung
carnitine dalam jumlah kecil. Kebanyakan buah, sayuran, dan biji-bijian
hampir tidak mengandung carnitine, kecualitempe dan alpukat, yang mengandungsejumlah kecil carnitine.
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Faktor Berpengaruh terhadap Carnitine
L-Carnitine bekerja paling efektif jika dikombinasikan dengandiet rendah gula dan karbohidrat.Carnitine dapat bekerja secara efektif, asupan karbohidrat
harus dijaga di bawah 50% dari total kalori yang dikonsumsisehari-hari.Peningkatan kadar insulin menghambat aktivitas carnitineyang optimal (pembakaran lemak tubuh dan produksi energi).Asam lemak Omega 3 (yang bisa didapat dari biji rami atauflaxseed dan minyak ikan) mampu meningkatkan aktivitasdan fungsi carnitine dalam tubuhMinyak biji rami atau flaxseed oil tidak hanya meningkatkanaktivitas carnitine di dalam tubuh, namun dapat menurunkan
risiko kematian akibat penyakit jantung koroner.Konsumsi protein yang cukup setiap kali makan sangatpenting untuk aktivitas carnitine dan pembakaran lemak.
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Manfaat L-Carnitine
membantu transportasi asam lemak rantai panjang yang dibakarmenjadi energi.
meningkatkan jumlah lemak yang dibakar tubuh baik selama
latihan anaerobik (latihan beban) maupun latihan aerobik (kardio)yang penting untuk menurunkan berat badan permanen.
berperan penting dalam memobilisasi timbunan lemak dalamjaringan tubuh yang kelebihan berat badan. membantu membuang limbah oksidasi lemak (keton) dari aliran
darah. Aktivitas carnitine yang efektif membantu mencegah lemak
menumpuk di hati, jantung, dan otot mampu meningkatkan kekuatan otot. memelihara jaringan otot membantu untuk menstabilkan gula darah dan menghilangkan
kecanduan karbohidrat (carbohydrate craving).
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Fatty acids mechanism in
mitochondria
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Carnitine deficiency syndromes
Haemodialysis Heart desease
Cardiac dysfunction
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Bacterial metabolisme
carnitineLysine
-Butyrobetaine
Crotonobetaine
L-Carnitine
Trimethylamine
Crotonobetaine reductase
L-Carnitine dehydratase
-Butyrobetaine hydroxylase
Monooxygenase
Monooxygenase
???Glycine
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L-Carnitine
D-Carnitine 3-Dehydrocarnitine
Glycine betaineGlycine
D-CarDehydrogenase
NAD+ NADH + H+
L-CarDehydrogenase
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Structure and function ofD-carnitine
dehydrogenase ofAgrobacteriumsp.525a
and enzymatic production ofL-carnitine
Screening ofD-carnitine dehydrogenase
producing microorganisms and optimisation ofD-carnitine dehydrogenase production
Purification and properties ofD-carnitine
dehydrogenase ofAgrobacteriumsp. 525a
Cloning and expression ofD-carnitinedehydrogenase
Conversion ofD-carnitine into L-carnitine by
partially purified enzymes
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Strategy for screening1. Isolation of D-carnitine utilizing bacteria
2. Determination of D-carnitine dehydrogenase activity (399)
The increase of absorbance at 340 nm was measured
spectrophotometrically at 30C
D-carnitine medium :D-carnitine HCl 5.0 g
K2HPO4 2.0 gMgSO4 7H2O 0.5 gYeast Extract 0.5 gDistilled water 1,000 mlpH was adjusted with 4.0 M NaOH to 7.0
Standard reaction mixture of D-carnitine dehydrogenase :Glycine-NaOH buffer (pH 9.5) 150 molD-Darnitine 22.5 mol-NAD+ 0.75 molCell-free extract 100-200 l
Total volume 1.5 ml
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continuous..
3. Evaluation of stability of D-carnitine dehydrogenase (48)
Evaluation of stability of D-carnitine dehydrogenase against dialysis
Effect of SH group protecting reagent on stability of D-carnitinedehydrogenase
Evaluation of stability of D-carnitine dehydrogenase against salting out
Evaluation of stability of D-carnitine dehydrogenase against preservation
4. Estimation af apparent Km values for D-carnitine and NAD+ (3)
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Taxonomic characteristic of an isolated
bacterium, 525a
Based on the characteristics as shown above, the strain isestimated to be Agrobacterium sp.
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Time course for the growth and production ofD-
carnitine dehydrogenase in Agrobacteriumsp. 525a
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Polyacrylamide gel electrophoresis and N-terminal
amino acid sequence of D-carnitine dehydrogenase
B. SDS-PAGEA. Native-PAGE
1. Polyacrylamide gel electrophoresis
1. Coomasive brilliant blue R-250staining
2. Activity staining3. Marker proteins4. Purified enzyme
2. N-terminal amino acid sequence of D-carnitine dehydrogenase5 10 15 20 25 30
M Q N L R R V L I T A A X S G I G R E I A K A F V N E G H L
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Estimation of the molecular weight of D-
carnitine dehydrogenase
Effect of temperature and pH on the
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Effect of temperature and pH on theoxidation and reduction of D-carnitine
dehydrogenase activity
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Effects of temperature and pH on the
stability of D-carnitine dehydrogenase
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Substrate specificity ofD-carnitine
dehydrogenase
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Double-reciprocal plots of initial
velocity ofD
-carnitine dehydrogenase
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Effect of metal ions and various reagents
on D-carnitine dehydrogenase activity
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Cloning strategy forD-carDH gene
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Preparation of synthetic primer
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Southern hybridization analysis of genomic
DNA from Agrobacteriumsp. 525a
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Cloning strategy for D-carDH
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Analysis of plasmid DNA
A. Agarose gel
1) /Sty I digest2) pDCP 3/Pst I digest
B. Southern hybridization of
pDCP 3/Pst I digest
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Expression ofD-carnitine dehydrogenase
from Agrobacteriumsp. 525a in E.coli
Host strain Plasmid Enzyme activity
(U/ml of culture)Agrobacteriumsp. 525a - 0.05
E.coliXL-1 blue pDCP 4 0.04
E.coliXL-1 blue pDCP 3 0.07
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Enzymatic production on L-carnitine
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Carnitine racemization on bacteria
Time course for the growth, D- and L-carDH
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activities and L-carnitine production on D-
carnitine medium
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Effect of pH and temperature on
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Effect of pH and temperature on
the L-carnitine production
Effect of NAD+ on the
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production of L-carnitine
Time course of L-carnitine production and
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the decrease in NAD+ during the conversion
reaction
Effect of metal ions and various
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Effect of metal ions and various
inhibitors on NAD+ degrading activity
Time course of L-carnitine production and
i ti l t d
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conversion reaction supplemented o-
phenanthroline
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Conclusion
One strain was selected by virtue of its productivity and
stability of the enzyme, and low Km value for D-
carnitine. An isolated strain was named to beAgrobacterium sp. 525 a
D-Carnitine dehydrogenase produced by Agrobacterium
sp. 525a was purified to apparent homogeneity. The
enzyme was purified 614 fold, giving a preparation witha specific activity of 30.7 units/mg protein. The N-
terminal 30 amino acids were sequenced. Enzymztic
properties of the enzyme were characterized. D-
Carnitine dehydrogenase is a new enzyme which has
not been described in the EC nomenclature.
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Cloning and expression of D-carnitine dehydrogenase gene
was done. The transformant which had the carnitine
dehydrogenase activity was isolated out of about 4,000 Ampr
colonies. One of two colonies was selected by high enzyme
activity. The isolated clone had about 3 kb fragment derived
from Agrobacterium sp. 525a gene.
Optimization of L-carnitine conversion reaction by partially
purified D- and L-carnitine dehydrogenase was done. O-Phenanthroline was found to protect the decrease of NAD in
the reaction mixture during conversion reaction. About 0.13 M
of D-carnitine was converted to L-carnitine under the following
reaction condition; 66.7 mM Tris-HCl buffer (pH 8.5), 0.3 M D-
carnitine, 5 mM NAD, 2 mM reduced glutathione, and 1 mM o-phenanthroline, at 30 C for 15 h. The conversion rate was
about 43%.
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Acknowledgments
Prof. Dr. Yutaka Kitamoto
Dr. Nobuhiro Mori Dr. Yoshiaki Yamano
Sasakawa Scientific Research grant
Hashitani foundation
Tottori University
All member of the laboratory of
Microbial Biotechnology
My wife and my children
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