Enzymes

In recent years, enzymes have gained wider applications in the biotechnology industry. The global industrial market has recently been valued at $1.4 billion, with an expected market increase of 4-5% (99). Enzymes are grouped into six major classes: oxidoreductases, transferases, hydrolases, lyases, ligases, and isomerases. The majority of commercialized enzymes are hydrolases such as amylase, cel-lulase, xylanases, pectinase, proteases, lipase, and collagenase. Other important enzymes are glucose isom-erase and glucose oxidase. Anaerobic bacteria are very diverse and thus produce a wide variety of enzymes, excluding oxygenases. Enzymes are widely used in pulp and paper, textile, detergent, and food- and feed-additive industries. In addition, enzymes also find applications in pharmaceutical synthesis, therapeutic contexts, and clinical and chemical analysis. The rumen anaerobic microbial population presents a rich and, until recently, underutilized source of novel enzymes with tremendous potential for industrial applications. The enzymes from these microorganisms include cellulases, xylanases, /-glucanases, pec-tinases, amylases, proteases, phytases, and tannases. High-molecular-mass complexes containing numerous cel-lulases have been identified in a number of rumen bacteria, including Butyrivibrio fibrisolvens, Ruminococcus al-bus, and Fibrobacter succinogenes.

The most common enzyme-producing anaerobic bacteria are mesophilic and thermophilic clostridia, and moderate- and hyper-thermophilic nonclostridial species (100,101). Among the amylolytic enzymes, a-amylase hy-drolyzes internal a-1,4 linkages of starch at random in an endo fashion, producing oligosaccharides of varying chain lengths. Generally, it cannot act on a-1,6 linkages of starch. C. butyricum, C. acetobutylicum, T. ethanolicus, C. ther-moamylolyticum have been reported to produce this amylase enzyme. /-Amylase hydrolyzes alternate a-1,4-glycosidic linkages of starch in an exo fashion from the nonreducing end, producing /-maltose. The /¡-amylase has been produced in high yield as a primary product during growth of Thermoanaerobacterium thermosulfurigenes (102). A hyperproductive mutant was isolated that produced eightfold more /-amylase than the wild type. Synthesis of the enzyme was both constitutive and resistant to catabolite repression. The /-amylase has also displayed industrial potential for the production of high-maltose syrups from raw or soluble starch at 75 °C. Various maltose-containing syrups are used in the brewing, baking, canning, and confectionery industries.

Glucoamylase is an exoacting carbohydrase that cleaves glucose units consecutively from the nonreducing end of starch molecules. High levels of a thermostable glucoa-mylase activity has been reported in crude extracts of T ethanolicus 39E (103), although it was purified and later described as an a-glucosidase activity. Glucoamylase is widely used in alcoholic fermentation of starchy materials and in the commercial production of glucose and high-glucose corn syrups. However, the current source of this enzyme is fungal. Pullulanase is a debranching enzyme that specifically cleaves a-1,6 linkages in starch, amylo-pectin, pullulan, and related oligosaccharides. It is generally used in combination with saccharifying amylases such as glucoamylase, fungal a-amylase, or fungal /-amylase for the production of various sugar syrups because it improves saccharification and yield (104). Hyun and Zeikus (103) found that T. ethanolicus produces highly thermoactive and thermostable cell-bound pullulanase. They also developed a hyperproductive mutant that displayed improved starch metabolism features. Pullulanase has also been reported to be produced by C. thermosaccharolyticum. Taking advantage of the high thermoactivity and acidoactivity of these pullulanases, it may be assumed that these enzymes might effectively replace a-amylase and pullulanase in both starch liquefaction and saccharification processes. a-Glucosidase hydrolyzes terminal nonreducing a-1,4-linked glucose residues of various substrates, releasing aD-glucose. It is generally considered to be maltase, but has a wide specificity, being able to cleave glucosides of non-sugars in addition to maltose, maltotriose, and other mal-tooligosaccharides, and to transfer a-D-glycosyl residues of maltose and a-D-glucosides to suitable acceptors.

a-Amylase, which is used in starch liquefaction, solu-bilizes a-1-4 linkages in starch-forming maltodextrin syrups. a-Amylase has been recently described in thermoan-aerobes. For example, the Pyrococcus furiosus a-amylase gene has been cloned and expressed in E. coli (105). The P. furiosus a-amylase is twice as active as Bacillus litchen-

formis commercial a-amylase, but does not require Ca2 + for stability at high temperature (i.e., >100 °C).

The cellulase system in both bacteria and fungi comprises three different classes of enzymes: (1) endo-1,4-/-glucanases; (2) exo-1,4-/-D-glucanases, including both 1,4-/-D-glucan cellobiohydrolases and 1,4-/-D-glucan glucohydrolases; and (3) 1,4-/-D-glucosidases, also referred as cellobiases. Several species of cellulolytic clostridia have been described in the literature. These include Clostridium cellobioparum, C. acetobutylicum, Clostridium cellulovor-ans, Clostridium stercorarium, and Clostridium thermo-cellum. Most of the work on industrial cellulases has been accomplished using aerobic fungal systems. However, due to the high specific-enzyme activity on the one hand and the high thermostability on the other, the cellulases from C. thermocellum have been considered for potential industrial utilization in direct alcohol fermentations, but not for saccharification per se (i.e., glucose production). Recently, endoglucanases have been used in laundry detergents.

Collagenases are endopeptidases that hydrolyze native, insoluble fibrous collagen. One of the anaerobic bacteria producing collagenase that has been extensively studied is C. histolyticum. The collagenase of C. histolyticum is available commercially. Other collagenase-producing anaerobic bacterial species include C. collagenovorans and C. proteo-lyticum (106). Pure collagenase can be applied as a sensitive probe for biosynthetic studies and sequence determinations. It is useful in prevention or cure of keloids. Collagenase is useful for the dispersal and dissociation of animal tissues in the laboratory. It is routinely used to separate cells from their parent tissues. C. histolyticum also produces an extracellular sulfhydryl proteolytic enzyme called clostripain (clostridiopeptidase). This enzyme possesses amidase, esterase, and proteolytic activity, which is directed toward the carboxyl peptide linkage of arginine (107).

Pectin-degrading enzymes are produced by a variety of microorganisms, including clostridia. These have been produced by C. aurantibutyricum, C. felsineum, C. multifer-mentas, C. roseum, and T. thermosulfurigenes. An active thermostable polygalactunonate hydrolase and pectin methylesterase have been produced by T. thermosulfuri-genes (108). These thermostable pectinolytic activities may have application in fruit juice clarification and for processing food or agricultural/forestry products.

Several xylose (glucose) isomerases have been isolated from thermoanaerobes including from Thermotoga neapol-itana (109). This enzyme is more stable and active than commercial enzymes. An alkaline phosphatase that is more stable and active than commercial calf enzyme has been purified and characterized from T. neapolitana (110). Table 3 provides examples of some selected enzymes of industrial importance from anaerobic extremophiles. This serves to illustrate the diversity of enzymes from anaerobes.

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