0026-8933/99/3305- $22.00 © 1999 åÄàä “ç‡Û͇ /Interperiodica” 0801 Molecular Biology, Vol. 33, No. 5, 1999, pp. 801–806. Translated from Molekulyarnaya Biologiya, Vol. 33, No. 5, 1999, pp. 906–911. Original Russian Text Copyright © 1999 by Berezina, Zverlov, Lunina, Chekanovskaya, Dubinina, Liebl, Velikodvorskaya. INTRODUCTION Thermotoga neapolitana is a Gram-positive anaer- obic extremely thermophilic bacterium with the opti- mal growth temperature 90°C. Huber and Stetter [1] first found bacteria of the genus Thermotoga in geo- thermal precipitation and hot springs of the Volcano Island, Italy. Two closely related species, Th. neapol- itana and Th. maritima, are obligatory heterotrophs growing on polymeric carbohydrates, starch in partic- ular [2]. Thermotoga amylases are highly thermo- stable and have a high specific activity, thus being of theoretical and industrial interest. The objectives of this work were to construct a Th. neapolitana genome library in Escherichia coli strain XL1-Blue, to select recombinant clones that express thermostable amylase, to sequence Th. nea- politana inserts of these clones, and to purify and to characterize their thermostable products. EXPERIMENTAL Bacterial strains and plasmids. We used Th. nea- politana Z2706-MC24, E. coli XL1-Blue [3], and pUC18 [4]. Reagents. We used trypton, yeast extract, bactoa- gar (Difco), soluble starch, amylose, amylopectin (Koch-Light), glycogen, pullulan, maltose, maltotri- ose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, isomaltose, panose, turanose, raffi- nose, melibiose, cellobiose, sucrose, glucose (Sigma). Enzymes were used as recommended by Promega, Amersham, and Boehringer Mannheim. DNA manipulations and sequencing. Chromo- somal DNA was isolated from Th. neapolitana culture grown in the Institute of Microbiology [5]. To construct a genome library, Th. neapolitana DNA was partly digested with Sau3A and ligated with pUC18. The product mixture was used to transform E. coli XL1-Blue. Isolation of plasmid DNA from E. coli, hydrolysis with restriction nucleases, ligation, agarose gel elec- trophoresis, transformation, Southern blot hybridiza- tion, and other manipulations were by the standard protocols [6]. Both strands of plasmid DNA were sequenced with a GATC Bio-Cycle sequencing kit (Konstanz) and biotinylated primers. DNA fragments were electro- phoretically separated in a GATC 1500 direct-blotting electrophoresis apparatus (Konstanz) and visualized with streptavidin-conjugated phosphatase and chromogenic substrate NBT-BCIP (Promega). The results were ana- lyzed with the DNASIS (Hitachi Software Engineering) and NCBI BLAST (http:www.ncbi.nlm.nih.gov) pro- grams. Detection of amylolytic activity in dishes. To identify clones producing thermostable amylase, cells were grown on 0.7% agar containing 0.5% substrate APPLIED MOLECULAR BIOLOGY Gene and Properties of Thermostable 4-a-Glucanotransferase of Thermotoga neapolitana O. V. Berezina 1 , V. V. Zverlov 1 , N. A. Lunina 1 , L. A. Chekanovskaya 1 , E. N. Dubinina 1 , W. Liebl 2 , and G. A. Velikodvorskaya 1 1 Institute of Molecular Genetics, Russian Academy of Medical Sciences, Moscow, 123182 Russia; E-mail: img@img.ras.ru 2 Institute of Microbiology and Genetics, George-August University, Goettingen, D-37077 Germany Received February 18, 1999 Abstract—Two clones that produced a thermostable enzyme hydrolyzing starch, amylose, and amylopectin were selected from a genome library of anaerobic extremely thermophilic bacterium Thermotoga neapolitana. The inserts of their recombinant plasmids were sequenced. Both plasmids had the mgtA gene for maltodextrin glycosyltransferase of 442 amino acid residues. The deduced molecular weight of the enzyme, 51.9 kDa, was confirmed by SDS-PAGE. Maltodextrin glycosyltransferase (4-α-glucanotransferase) [EC 2.4.1.25] hydro- lyzed the 1,4-α-glycoside bonds in oligomeric and polymeric 1,4-α-glucans and transferred oligosaccharides (maltotriose being the shortest one) to acceptor maltodextrins. The highest enzymic activity was observed at pH 7.0 and at 85°C. Key words: Thermotoga neapolitana, 4-α-glucanotransferase, thermostable amylolytic enzymes, substrate specificity, nucleotide sequence UDC 577.29:577.112.083.3