Ochrobactrum L-3的L-肉碱脱氢酶研究
发布时间：2018-09-08 21:42
【摘要】： L-肉毒碱(L-Camitine,CN)又叫L-肉碱、维生素B_T、左卡尼汀,化学名称为L-β-羟基-γ-三甲胺丁酸。L-肉碱是一种基本的细胞成分.主要位于线粒体内膜上,是长链脂肪酸进入线粒体的运输载体。自从1973年Engel报告第一例人类CN缺乏症(CD)以来,有关L-肉碱的生理生化,CD的病理和临床报告日渐增多,成为临床医学和营养学的一个重要课题。L-肉碱作为药物治疗由CD引起的各种疾病取得了很好的效果,其生产方法的改进和创新对于L-肉碱的大规模生产具有重要意义。目前生产L-肉碱的方法中,酶转化法具有环境友好、成本低、产品安全性高等特点,是较有吸引力的方法。 在酶转化法中,L-肉碱脱氢酶(EC 1.1.1.108)是所需要的一个重要的酶,因此它的性质对该方法的使用和推广非常重要。虽然已有少量文献对此类酶进行了报道,但在这些已报道的酶中,大多数都存在着酶活和专一性不高的问题。 本研究从土壤中分离筛选到一株产L-肉碱脱氢酶菌株L-3,对其进行了系统地分类学鉴定,并研究了该菌株产L-肉碱脱氢酶的发酵条件,进行了该酶的纯化和对酶性质研究。主要实验结果如下: 1.菌株的筛选分离及鉴定经过富集培养划线分离纯化后,得到了一株能够生产L-肉碱脱氢酶的菌株,通过对该菌株的形态学研究,生理生化测定,16SrDNA测序和系统发育分析,确定菌株L-3属于苍白杆菌属(Ochrobactrum sp)。 2、菌株产酶发酵研究对菌株L-3产酶发酵的条件进行了研究。研究采用摇瓶培养试验,以测定菌体生长量、酶活性为指标,确定了该菌株产酶发酵的最佳条件为,培养基组成:0.5%L-肉碱,0.2%葡萄糖,0.2%K_2HPO_4·3H_2O,0.05%KH_2PO_4,0.05%MgSO_4·7H_2O,1.0%(v/v)微量元素(包括5.2%ZnSO_4,5.0%FeSO_4·7H_2O,5.0%CuSO_4·7H_2O和0.05%MnSO_4·H_2O),pH 6.5。培养温度30℃,装液量为100 mL三角瓶装20 mL培养基,于120 rpm转速下培养24 h。在上述培养条件下发酵,发酵液的酶活可达1.46U/ml。 3.酶的纯化和性质研究在发酵罐中培养10L菌液,离心收集,沉淀经超声波破壁后得到粗酶液。粗酶液经硫酸铵分级沉淀、DEAE-Cellulose离子交换层析、Toyopearl Hw一65C疏水层析、Sephadex G一75分子筛层析后,纯度提高约51倍,比活达到2.55U/mg。聚丙烯酰胺凝胶电泳(SDS-PAGE)显示为单一条带,分子量约为57kD。研究L一肉碱脱氢酶的特性得知:该酶反应的最适pH为9.5;最适作用温度为50~C:在pH6.0~11.0、55℃以下稳定;对L-肉碱的Km值为5.9mmol/L,转化系数Kcat为3.4/s:Hg2',pb2+等几乎使该酶完全失活。
[Abstract]:L-carnitine (L-carnitine), also called L-carnitine, vitamin B, levacarnitine, chemical name L- 尾-hydroxy- 纬-trimethylaminobutyric acid. L- carnitine is a basic cell component. It is mainly located on the inner membrane of mitochondria and is the transport carrier of long chain fatty acids into mitochondria. Since Engel reported the first human CN deficiency (CD) in 1973, the number of pathological and clinical reports on L- carnitine physiological and biochemical CD has been increasing. L- carnitine has been used as a drug to treat various diseases caused by CD. The improvement and innovation of its production method is of great significance for the large-scale production of L-carnitine. Enzyme conversion is an attractive method for producing L-carnitine because of its environmental friendliness, low cost and high product safety. L- carnitine dehydrogenase (EC 1.1.1.108) is an important enzyme needed in enzymatic transformation, so its properties are very important for the application and popularization of this method. Although there have been a few reports on these enzymes, most of them have the problem of low activity and specificity. In this study, a strain L-3 producing L-carnitine dehydrogenase was isolated and screened from soil. It was systematically identified by taxonomy, and the fermentation conditions of L-carnitine dehydrogenase were studied. The purification and properties of L-carnitine dehydrogenase were studied. The main results are as follows: 1. A strain capable of producing L-carnitine dehydrogenase was obtained after screening, isolation, identification, enrichment, culture, and purification. The strain was characterized by morphological study, physiological and biochemical analysis, sequencing and phylogenetic analysis of 16s rDNA. Strain L-3 belongs to the genus (Ochrobactrum sp). _ 2. The fermentation conditions of strain L-3 were studied. The optimum conditions for enzyme fermentation of this strain were determined by using shaking flask culture test. The optimum conditions for enzyme fermentation of the strain were determined as follows: the composition of the medium was: 1: 0.5L- 0.2L- carnitine 0.2HPO-0.2H2O-0.5KH2PO40.05KH2PO40.05MgSO47H2O2O1.0% (vrv) trace elements (including 5.2ZnSO4, 5.0SO_4 7H2O-5.0CuSO4 7H_2O and 0.05%MnSO_4 H2O). The culture temperature was 30 鈩，
本文编号：2231777
[Abstract]:L-carnitine (L-carnitine), also called L-carnitine, vitamin B, levacarnitine, chemical name L- 尾-hydroxy- 纬-trimethylaminobutyric acid. L- carnitine is a basic cell component. It is mainly located on the inner membrane of mitochondria and is the transport carrier of long chain fatty acids into mitochondria. Since Engel reported the first human CN deficiency (CD) in 1973, the number of pathological and clinical reports on L- carnitine physiological and biochemical CD has been increasing. L- carnitine has been used as a drug to treat various diseases caused by CD. The improvement and innovation of its production method is of great significance for the large-scale production of L-carnitine. Enzyme conversion is an attractive method for producing L-carnitine because of its environmental friendliness, low cost and high product safety. L- carnitine dehydrogenase (EC 1.1.1.108) is an important enzyme needed in enzymatic transformation, so its properties are very important for the application and popularization of this method. Although there have been a few reports on these enzymes, most of them have the problem of low activity and specificity. In this study, a strain L-3 producing L-carnitine dehydrogenase was isolated and screened from soil. It was systematically identified by taxonomy, and the fermentation conditions of L-carnitine dehydrogenase were studied. The purification and properties of L-carnitine dehydrogenase were studied. The main results are as follows: 1. A strain capable of producing L-carnitine dehydrogenase was obtained after screening, isolation, identification, enrichment, culture, and purification. The strain was characterized by morphological study, physiological and biochemical analysis, sequencing and phylogenetic analysis of 16s rDNA. Strain L-3 belongs to the genus (Ochrobactrum sp). _ 2. The fermentation conditions of strain L-3 were studied. The optimum conditions for enzyme fermentation of this strain were determined by using shaking flask culture test. The optimum conditions for enzyme fermentation of the strain were determined as follows: the composition of the medium was: 1: 0.5L- 0.2L- carnitine 0.2HPO-0.2H2O-0.5KH2PO40.05KH2PO40.05MgSO47H2O2O1.0% (vrv) trace elements (including 5.2ZnSO4, 5.0SO_4 7H2O-5.0CuSO4 7H_2O and 0.05%MnSO_4 H2O). The culture temperature was 30 鈩，
本文编号：2231777
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