Transcript
  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    1/47

    @siswa setyahadi

    D-Carnitine Dehydrogenase dari

    Agrobacteriumsp. 525a

    Siswa SetyahadiPusat Teknologi BioindustriDeputi Teknologi Agroindustri dan Bioteknologi,

    Badan Pengkajian dan Penerapan Teknologi

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    2/47

    @siswa setyahadi

    http://www.sportindo.com/products/l-carnitine
  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    3/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    4/47

    @siswa setyahadi

    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.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    5/47

    @siswa setyahadi

    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.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    6/47

    @siswa setyahadi

    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.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    7/47

    @siswa setyahadi

    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.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    8/47

    @siswa setyahadi

    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).

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    9/47

    @siswa setyahadi

    Fatty acids mechanism in

    mitochondria

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    10/47

    @siswa setyahadi

    Carnitine deficiency syndromes

    Haemodialysis Heart desease

    Cardiac dysfunction

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    11/47

    @siswa setyahadi

    Bacterial metabolisme

    carnitineLysine

    -Butyrobetaine

    Crotonobetaine

    L-Carnitine

    Trimethylamine

    Crotonobetaine reductase

    L-Carnitine dehydratase

    -Butyrobetaine hydroxylase

    Monooxygenase

    Monooxygenase

    ???Glycine

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    12/47

    @siswa setyahadi

    L-Carnitine

    D-Carnitine 3-Dehydrocarnitine

    Glycine betaineGlycine

    D-CarDehydrogenase

    NAD+ NADH + H+

    L-CarDehydrogenase

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    13/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    14/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    15/47

    @siswa setyahadi

    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)

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    16/47

    @siswa setyahadi

    Taxonomic characteristic of an isolated

    bacterium, 525a

    Based on the characteristics as shown above, the strain isestimated to be Agrobacterium sp.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    17/47

    @siswa setyahadi

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    18/47

    @siswa setyahadi

    Time course for the growth and production ofD-

    carnitine dehydrogenase in Agrobacteriumsp. 525a

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    19/47

    @siswa setyahadi

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    20/47

    @siswa setyahadi

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    21/47

    @siswa setyahadi

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    22/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    23/47

    @siswa setyahadi

    Estimation of the molecular weight of D-

    carnitine dehydrogenase

    Effect of temperature and pH on the

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    24/47

    @siswa setyahadi

    Effect of temperature and pH on theoxidation and reduction of D-carnitine

    dehydrogenase activity

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    25/47

    @siswa setyahadi

    Effects of temperature and pH on the

    stability of D-carnitine dehydrogenase

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    26/47

    @siswa setyahadi

    Substrate specificity ofD-carnitine

    dehydrogenase

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    27/47

    @siswa setyahadi

    Double-reciprocal plots of initial

    velocity ofD

    -carnitine dehydrogenase

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    28/47

    @siswa setyahadi

    Effect of metal ions and various reagents

    on D-carnitine dehydrogenase activity

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    29/47

    @siswa setyahadi

    Cloning strategy forD-carDH gene

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    30/47

    @siswa setyahadi

    Preparation of synthetic primer

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    31/47

    @siswa setyahadi

    Southern hybridization analysis of genomic

    DNA from Agrobacteriumsp. 525a

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    32/47

    @siswa setyahadi

    Cloning strategy for D-carDH

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    33/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    34/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    35/47

    @siswa setyahadi

    Enzymatic production on L-carnitine

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    36/47

    @siswa setyahadi

    Carnitine racemization on bacteria

    Time course for the growth, D- and L-carDH

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    37/47

    @siswa setyahadi

    activities and L-carnitine production on D-

    carnitine medium

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    38/47

    @siswa setyahadi

    Effect of pH and temperature on

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    39/47

    @siswa setyahadi

    Effect of pH and temperature on

    the L-carnitine production

    Effect of NAD+ on the

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    40/47

    @siswa setyahadi

    production of L-carnitine

    Time course of L-carnitine production and

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    41/47

    @siswa setyahadi

    the decrease in NAD+ during the conversion

    reaction

    Effect of metal ions and various

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    42/47

    @siswa setyahadi

    Effect of metal ions and various

    inhibitors on NAD+ degrading activity

    Time course of L-carnitine production and

    i ti l t d

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    43/47

    @siswa setyahadi

    conversion reaction supplemented o-

    phenanthroline

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    44/47

    @siswa setyahadi

    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.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    45/47

    @siswa setyahadi

    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%.

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    46/47

    @siswa setyahadi

    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

  • 7/30/2019 Materi Kuliah Pemanfaatan Biokimia (Carnitine Dehydrogenase)

    47/47

    @siswa setyahadi


Recommended