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Study of L-carnitine Dehydrogenase Form Ochrobactrum L-3

Author: WangLei
Tutor: MaXiaoHang；JiaXiaoMing
School: Zhejiang University
Course: Microbiology
Keywords: Carnitine L-carnitine dehydrogenase Ochrobactrum
CLC: R341
Type: Master's thesis
Year: 2008
Downloads: 59
Quote: 1
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Abstract

Camitine, namely 3-hydroxy-4-trimethylaminobutyrate,VBT.Carnitine is a basic component in cell, which is mainly located at the cell membrane and functions as a vector in the transport of long-chain fatty acids through the inner itochondrial membrane. Since the first case of camitine deficiency in human reported by Engel in 1973, more physiological and biochemical properties as well as clinical cases and pathology of camitine deficiency has been increasingly reported, and it has become an important subject in clinical medicine and nutriology. Camitine has a good effect on the treatment of diseases caused by CD. Thus, it is important to improve the process for the large scale production of camitine. Among all the mehods of canitine production, enzymic transformation has such advantages as environmentally friendly, low cost and high level of safety, whch is a very appealing method.The L-camitine dehydrogenase （EC 1.1.1.108 ） is a important enzyme to be used in the enzymic transformation method. Consequently, the properties of it were essential for the practical application in the enzymic transformation. At present, although some researches of L-camitine dehydrogenase had been reported, in most cases, the enzymes’ characteristics are not very satisfied.In this study, a strain of bacteria capable of producing L-camitine dehydrogenase was isolated and it was identified subsequently as a strain of the genus Ochrobactrum. In this work the conditions of the production, as well as the characteristics of the enzyme were studied. The results were showed as follows:1. The isolation and identification of L-camitine dehydrogenase producing strain: A bacterium which can produce L-camitine dehydrogenase was isolated after enrichment culture and lineation purification. Based on its morphological studies, physiochemical identification, and 16S rDNA sequencing and phylogenetic analysis, this isolate was indentified to be a Ochmbactrum.sp2. The enzyme fermentation conditions: Based on the studies for the conditions of enzyme production, the optimum fermentation conditions were established. The medium was composed of the following ingredients: 0.5% Carnitine, 0.2% Glucose, 0.2% K₂HPO₄·3H₂O, 0.05% KH₂PO₄, 0.05% MgSO₄-7H₂O, 1.0% （v/v） trace elements solution containing of 5.2% ZnSO₄, 5.0% FeSO₄·7H₂O, 5.0% CuSO₄·7H₂O and 0.05% MnSO₄·H₂O, pH 6.5. When the bacterium was inoculated in a 100 ml flask containing 20 ml medium and incubated on a shaker of 120 rpm at 30℃for 24 h, the L-carnitine dehydrogenase activity peaked at 1.46U/ml.3. Purification and characteristics of L-carnitine dehydrogenase: After cultivation, cells were harvested from 10 L culture medium by centrifugation and the deposition was disrupted with a ultrasonic device to obtain crude enzyme. The enzyme was purified 51 folds after gradient precipitation with （NH₄）₂SO₄, DEAE-Cellulose ion exchange chromatography, Toyopearl HW-65C hydrophobic chromatography and Sephadex G-75 molecular-exclusion chromatography. The purified enzyme was homogeneous as judged by PAGE, and the subunit molecular mass was estimated to be 57 KDa by SDS-PAGE.Characteristics studies showed that the optimal reaction for the enzyme was 50℃and the optimal pH was 6.5. It was stable in the pH range of 6. 0～11.0 and at the temperature below 55℃. The Km value for L-carnitine dehydrogenase is 5.9 mmol/1 and transformation rate （Kcat） is34/s. The enzyme was markedly inactivated by Hg²⁺ ,Pb²⁺ et al.

Study of L-carnitine Dehydrogenase Form Ochrobactrum L-3

Abstract

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