Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli

Abstract

Clostridium thermohydrosulfuricum 39E, a gram-positive thermophilic anaerobic bacterium, produced a cyclodextrin (CD)-degrading enzyme, cyclodextrinase (CDase) (EC 3.2.1.54). The enzyme was purified to homogeneity from Escherichia coli cells carrying a recombinant multicopy plasmid that contained the gene encoding for thermophilic CDase. The purified enzyme was a monomer with an Mr of 66,000 ± 2,000. It showed the highest activity at pH 5.9 and 65°C. The enzyme hydrolyzed a-, j3-, and 'y-CD and linear maltooligosaccharides to yield maltose and glucose. The Km values for a-, 0-, and fy-CD were 2.5, 2.1, and 1.3 mM, respectively. The rates of hydrolysis for polysaccharides (starch, amylose, amylopectin, and pullulan) were less than 5% of the rate of hydrolysis for a-CD. The entire nucleotide sequence of the CDase gene was determined. The deduced amino acid sequence of CDase, consisting of 574 amino acids, showed some similarities with those of various amylolytic enzymes.Cyclodextrins (CDs) are cyclic oligosaccharides consisting of a-1,4-linked 6-, 7-, or 8-glucopyranose units usually referred to as a-, 1-, or -y-CDs, respectively. One of the remarkable properties of the CDs is their resistance to hydrolysis by the common starch-splitting enzymes. Among the bacterial amylases, only those from Bacillus subtilis (27), Pseudomonas sp. (14), and Flavobacterium sp. (2) catalyze the hydrolysis of CDs; for the first of these enzymes, CDs are poor substrates, while the latter two enzymes degrade CDs faster than starch. Until now, only four strains, B. macerans (7), B. coagulans (16), B. sphaericus (29), and a Bacillus sp. (43), have been known to produce cyclomaltodextrinase (EC 3.2.1.54, cyclomaltodextrin hydrolase, decycling) (CDase), an enzyme which effectively hydrolyzes CDs and linear maltodextrins. In contrast to the extensive information concerning amylolytic enzymes, studies on CDases have been confined to these four enzymes. To our best knowledge, nothing to date has been reported about the gene structure of CDases.In the course of our studies on thermophilic bacteria, we recently characterized a thermophilic CDase from Clostnidium thermohydrosulfuricum 39E (33). This enzyme was partially purified (about 205-fold), and many of its properties were determined. However, because the CDase was not purified to homogeneity, important molecular and kinetic properties of the enzyme remain unknown. In the present study, we report on the purification and detailed characterization of C thermohydrosulfuricum 39E CDase from Esch-

A New Thermoactive Pullulanase from Desulfurococcus mucosus : Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis

“…Although more detailed studies are required to determine the number of the active sites present in a protein, our results are indicative of competition between pullulan and amylose, suggesting the presence of a single active site capable of hydrolysis of both types of linkages. In addition, linear sequence alignment with the cyclodextrinase of Thermoanaerobacter ethanolicus (formerly Clostridium thermohydrosulfuricum 39E) (37), which possesses one catalytic site, showed clearly that the three catalytic residues, Asp-325, Glu-354, and Asp-421, were absolutely conserved in the D. mucosus pullulanase sequence at positions Asp-363, Glu-394, and Asp-461.…”

Expression of periplasmic α-amylase of Xanthomonas campestris K-11151 in Escherichia coli and its action on maltose

“…6b), which can also act on starch-related materials, pullulan and CDs (Brumm etal., 1991a), but has a slightly different substrate specificity in that the rate for a-CD is less than 20% of those for B-and y-CDs, and glycogen is a better substrate than pullulan, amylose and amylopectin. The primary sequence of the periplasmic aamylase from x. campestris K-11151 showed a low degree of identity with cyclodextrinase from Tbermoanaerobacter etbataoliczls (Podkovyrov & Zeikus, 1992) (18 % ; Fig. 6c) and neopullulanase from Bacill~s stearotbermophilzis (Kuriki & Imanaka, 1989) (19 % ; Fig.…”

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

“…Cyclomaltodextrinase (CDase) [EC 3.2.1.54; cyclomaltodextrin dextrin-hydrolase (decyclizing)] is a unique enzyme which can hydrolyze CD and release the substance from CD inclusion complexes. After the first report of the CDase from Bacillus maceranse (8), CDases from various microorganisms such as Bacillus coagulans (23), Bacillus sphaericus E-244 (34,35), Clostridium thermohydrosulfuricum strain 39E (recently reclassified as Thermoanaerobacter ethanolicus strain 39E) (38,40), alkalophilic Bacillus sp. (50), Bacillus subtilis strain H-17 (24,25), Flavobacterium sp.…”