【摘要】： L-肉毒堿(L-Camitine,CN)又叫L-肉堿、維生素B_T、左卡尼汀,化學(xué)名稱為L(zhǎng)-β-羥基-γ-三甲胺丁酸。L-肉堿是一種基本的細(xì)胞成分.主要位于線粒體內(nèi)膜上,是長(zhǎng)鏈脂肪酸進(jìn)入線粒體的運(yùn)輸載體。自從1973年Engel報(bào)告第一例人類CN缺乏癥(CD)以來(lái),有關(guān)L-肉堿的生理生化,CD的病理和臨床報(bào)告日漸增多,成為臨床醫(yī)學(xué)和營(yíng)養(yǎng)學(xué)的一個(gè)重要課題。L-肉堿作為藥物治療由CD引起的各種疾病取得了很好的效果,其生產(chǎn)方法的改進(jìn)和創(chuàng)新對(duì)于L-肉堿的大規(guī)模生產(chǎn)具有重要意義。目前生產(chǎn)L-肉堿的方法中,酶轉(zhuǎn)化法具有環(huán)境友好、成本低、產(chǎn)品安全性高等特點(diǎn),是較有吸引力的方法。 在酶轉(zhuǎn)化法中,L-肉堿脫氫酶(EC 1.1.1.108)是所需要的一個(gè)重要的酶,因此它的性質(zhì)對(duì)該方法的使用和推廣非常重要。雖然已有少量文獻(xiàn)對(duì)此類酶進(jìn)行了報(bào)道,但在這些已報(bào)道的酶中,大多數(shù)都存在著酶活和專一性不高的問(wèn)題。 本研究從土壤中分離篩選到一株產(chǎn)L-肉堿脫氫酶菌株L-3,對(duì)其進(jìn)行了系統(tǒng)地分類學(xué)鑒定,并研究了該菌株產(chǎn)L-肉堿脫氫酶的發(fā)酵條件,進(jìn)行了該酶的純化和對(duì)酶性質(zhì)研究。主要實(shí)驗(yàn)結(jié)果如下: 1.菌株的篩選分離及鑒定經(jīng)過(guò)富集培養(yǎng)劃線分離純化后,得到了一株能夠生產(chǎn)L-肉堿脫氫酶的菌株,通過(guò)對(duì)該菌株的形態(tài)學(xué)研究,生理生化測(cè)定,16SrDNA測(cè)序和系統(tǒng)發(fā)育分析,確定菌株L-3屬于蒼白桿菌屬(Ochrobactrum sp)。 2、菌株產(chǎn)酶發(fā)酵研究對(duì)菌株L-3產(chǎn)酶發(fā)酵的條件進(jìn)行了研究。研究采用搖瓶培養(yǎng)試驗(yàn),以測(cè)定菌體生長(zhǎng)量、酶活性為指標(biāo),確定了該菌株產(chǎn)酶發(fā)酵的最佳條件為,培養(yǎng)基組成:0.5%L-肉堿,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。培養(yǎng)溫度30℃,裝液量為100 mL三角瓶裝20 mL培養(yǎng)基,于120 rpm轉(zhuǎn)速下培養(yǎng)24 h。在上述培養(yǎng)條件下發(fā)酵,發(fā)酵液的酶活可達(dá)1.46U/ml。 3.酶的純化和性質(zhì)研究在發(fā)酵罐中培養(yǎng)10L菌液,離心收集,沉淀經(jīng)超聲波破壁后得到粗酶液。粗酶液經(jīng)硫酸銨分級(jí)沉淀、DEAE-Cellulose離子交換層析、Toyopearl Hw一65C疏水層析、Sephadex G一75分子篩層析后,純度提高約51倍,比活達(dá)到2.55U/mg。聚丙烯酰胺凝膠電泳(SDS-PAGE)顯示為單一條帶,分子量約為57kD。研究L一肉堿脫氫酶的特性得知:該酶反應(yīng)的最適pH為9.5;最適作用溫度為50~C:在pH6.0~11.0、55℃以下穩(wěn)定;對(duì)L-肉堿的Km值為5.9mmol/L,轉(zhuǎn)化系數(shù)Kcat為3.4/s:Hg2',pb2+等幾乎使該酶完全失活。
[Abstract]:L-carnitine (L-carnitine), also called L-carnitine, vitamin B, levacarnitine, chemical name L- 尾-hydroxy- 緯-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，

本文編號(hào)：2231777