Amylolytic Enzymes and Solventogenic Clostridia

The solventogenic clostridia, like all clostridia, are Gram positive, spore forming, obligate anaerobes. These bacteria can change to a variety of morphologies during fermentation, with motile rod-shaped cells present during the exponential growth phase and dormant oval-shaped endospores formed when the culture encounters adverse conditions. The maintenance of cellular growth (like other heterotrophic bacteria) and butanol production by solventogenic clostridia depends on the utilization of nutrients obtained from the surroundings. Corn is principally com­posed of starch, and starch is made up of amylose and amylopectin. Amylose is composed of a linear polymer of glucose with links exclusively in the a-1, 4 orientation. On the other hand, amylopectin is a highly branched polysaccharide consisting of linear chains of a-1, 4-linked D-glucose residues, joined by a-1, 6-glucosidic bonds. The branch points occur on the average of every 20-25 D — glucose units, so that amylopectin contains 4-5% of a-1, 6-glucosidic linkages (Jensen and Norman, 1984). High-molecular-weight macromolecules like starch from corn are too large to be assimilated by the bacterial cells and therefore need to be hydrolyzed into low-molecular-weight products by specific extracellular depolymerases, which can then be taken into the cells via specific transport systems. Solventogenic clostridia have the ability to utilize a wide spectrum of carbohydrates through the secretion of several extracellular amylolytic enzymes.

Several amylolytic enzymes with different modes of action necessary for efficient and complete breakdown of starch to glucose have been identified in the solventogenic clostridia. They include a-amylase, p-amylase, glucoamylase, a — glucosidase, and pullulanase, and their mode of action and linkages hydrolyzed in the starch molecule and products formed are summarized in Table 6.1.

Amylases are enzymes that act on starch, glycogen, and derived polysaccha­rides. They hydrolyze a-1, 4 or a-1, 6 glucosidic bonds between consecutive glucose units. a-Amylase (1,4-a-D-glucanohydrolase; EC 3.2.1.1) catalyzes the hydrolysis of a-1,4 glucosidic bonds in the interior of the substrate molecule (starch, glycogen and various oligosaccharides) and produces a mixture of glu­cose, maltose, maltotriose, maltotetraose, maltopentose, maltohexaose, and oli­gosaccharides in a ratio depending on the source of the enzyme (Ezeji, 2001). The p-amylase (1, 4-a-D-glucan maltohydrolase; EC 3.2.1.2) hydrolyzes a-1,4 glucosidic bonds in starch and oligosaccharides producing maltose units from

the nonreducing terminal end of the substrate. Glucoamylase (1, 4-a-D-glucan glucohydrolase; EC 3.2.1.3) hydrolyzes both a-1, 4 and a-1, 6 glucosidic linkages from the nonreducing terminal end of the glucose units in the starch molecule. a-Glucosidase (a-D-glucoside glucohydrolase; EC 3.2.1.20) catalyzes, like glu­coamylase, the hydrolysis of the terminal nonreducing a-1, 4-linked glucose units in the starch. The preferred substrates for a-glucosidases are maltose, maltotriose, maltotetraose, and short oligosaccharides. Furthermore, pullulanases (a-dextrin 6-glucanohydrolase; EC 3.2.1.41) are enzymes that cleave -1, 6 linkages in pullulan and release maltotriose, although pullulan itself may not be the natural substrate.

Synergistic action between pullulanase and a-amylase enzymes of C. ther — mosulfurogenes has been demonstrated (Spreinat and Antranikian, 1992) and an a-glucosidase of C. beijerinckii has been shown to hydrolyze both types of glucosyl linkages (a-1, 4 and a-1, 6) (Albasheri and Mitchell, 1995). In addition, Paquet et al. (1991) purified and characterized novel C. acetobutylicum 824 a — amylase, which possesses some glucoamylase activity (2.7%).