CLONING, NUCLEOTIDE-SEQUENCES, AND ENZYMATIC-PROPERTIES OF GLUCOSE-DEHYDROGENASE ISOZYMES FROM BACILLUS-MEGATERIUM IAM1030
CLONING, NUCLEOTIDE-SEQUENCES, AND ENZYMATIC-PROPERTIES OF GLUCOSE-DEHYDROGENASE ISOZYMES FROM BACILLUS-MEGATERIUM IAM1030
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DOI：
10.1128/jb.174.15.5013-5020.1992
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发表时间：
1992-08-01
影响因子：
3.2
通讯作者：
OKADA, H
中科院分区：
文献类型：
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作者：
NAGAO, T;MITAMURA, T;OKADA, H
巨大芽孢杆菌已知有几个基因编码葡萄糖脱氢酶同工酶。其中GDHI和GDHII是我们在前期工作中从巨大芽孢杆菌IAM1030(T.Mitamura，R.V.Evora，T.Nakai，Y.Makino，S.Negoro，I.Urabe，H.Okada，J.Ferment)中克隆的。比昂斯。70：363-369,1990)。在本研究中，从同一菌株中分离到两个新基因gdhIII和gdhIV，并对它们的核苷酸序列进行了鉴定。每个基因都有一个783bp的开放阅读框，可编码261个氨基酸的多肽。因此，从巨大芽孢杆菌IAM1030中共克隆了四个葡萄糖脱氢酶基因。此外，该菌株似乎没有其他葡萄糖脱氢酶基因，可以与迄今通过Southern杂交分析检测的四个克隆基因区分开来。将新克隆的两个基因在大肠杆菌中表达，并对其产物GlcDH-III和GlcDH-IV进行了纯化和鉴定，并与GDHI和gdhII编码的另两种同工酶GlcDH-I和GlcDH-II进行了比较。这些同工酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(GlcDH-I和GT；GlcDH-III=GlcDH-II和GT；GlcDH-II)中表现出不同的迁移率，尽管它们的氨基酸残基相同。双向免疫扩散试验表明，GlcDH-I在免疫学上与其他同工酶不同，GlcDH-III和GlcDH-IV完全相同，但与GlcDH-II略有不同。这些葡萄糖脱氢酶在2M的氯化钠存在下稳定。氯化钠对最不稳定的谷氨酰胺脱氢酶-III的影响尤其大。动力学研究表明，虽然这些同工酶可以利用NAD和NADP，但根据辅酶的专一性，它们被分为两类：GlcDH-III和GlcDH-IV偏好NAD，GlcDH-I和GlcDH-II偏好NADP。并对这些葡萄糖脱氢酶基因的系统发育关系进行了讨论。
Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I > GlcDH-III = GlcDH-II > GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is the most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.