Heterologous Enzyme Production

The use of enzymes in the baking industry is widely extended. They modify the flour components, mainly starch, nonstarch polysaccharides, proteins, and lipids, and as a result, the physicochemical properties of the dough and the bread quality parameters (22).

Regular enzyme application in the dough system is carried out by exogenous addition of commercial preparations. Although substantial amounts of these compounds are obtained from recombinant organisms, mainly fungi and bacteria, all of them contain a cocktail of minor enzymatic activities that may negatively affect bread quality. Moreover, these products may act as allergens, producing a high prevalence of occupational hypersensitivity, in terms of dermal and bronchial (asthma) allergies (164-166). Alternatively, recombinant baker's yeast strains producing a technological enzyme can be employed to obtain the same benefits, avoiding airborne allergen pollution at the workplace (167).

11.4.1 Amylases

The amount of fermentable sugars present in flour is only around 1-2% (168). This is clearly insufficient for the optimum growth and gas production of yeast. The fermentation depends thus on the proper production of maltose from damaged starch by the action of wheat and a-amylases (169). Regular nongerminated wheat flour contains good levels of ^-amylase but little amount of a-amylases. Because of this, wheat flour is normally supplemented with exogenous a-amylase (170).

The reducing sugars produced by the action of a-amylases not only supply the yeast with the substrate for the proper production of gas, but they serve to improve the aroma and color of bread. Furthermore, the addition of a-amylase enhances bread volume, which results in a softer crumb (169,171).

Baker's yeast strains expressing the a-amylase from Aspergillus oryzae have been constructed (172). The a-amylase cDNA was placed under the control of the yeast ACT1 promoter and the resulting transformant produced a recombinant a-amylase with biochemical and kinetic characteristics similar to the native enzyme (173). Dough produced with this transformant gave bread with greater volume, a softer crumb, and a reduced firming rate (172). In addition, the authors showed that a fraction of the enzyme was somehow protected by the yeast structure, so it could remain active for a little bit longer in the oven, as an encapsulated enzyme, resembling then the action of a-amylases of intermediate thermal stability. Therefore, the action of a particular enzyme on its substrate may be modified just by being expressed by yeast.

11.4.2 Xylanases

Wheat flour contains up to 3% nonstarch polysaccharides (pentosanes or hemicellulos-es), which play an important role in water absorption and dough rheology. Enzymatic modification of these polysaccharides improves both dough properties and bread quality, increasing volume and retarding the process of "staling" during storage (22, 169).

Using the same strategy as previously described, Monfort et al. (141) expressed different xylanolytic enzymes in industrial baker's yeasts. When transformants producing the A. nidulans endoxylanase X24 (xlnB) were used in regular straight dough, only a discrete increase in bread volume (5%) was observed. However, when this enzyme and the A. oryzae a-amylase were coproduced simultaneously (174), a synergistic effect was observed. The bread elaborated with the new transformant showed a larger volume increase (around 30%) and a softer crumb texture than those prepared with either the xlnB overproducing transformant or a control strain. Thus, the combination of xylanase and fungal amylase provided a better overall score on dough and bread quality parameters for the combined exogenous addition of these enzymes (175).

11.4.3 Lipases

Lipases (glycerol ester hydrolases EC 3.1.1.3) catalyze the hydrolysis of triacylglycerols to render mono- and diacylglycerides and free fatty acids. The addition of lipases to the dough improves its rheological properties and stability, resulting in breads with larger volume, softer crumb, and longer shelf life (176). These effects depend on the type of flour and the baking formula, being significant in dough systems without added shortening (175). It has been postulated that the lipase activity would form a kind of in situ emulsion by producing mono and diglycerides into the dough (176). However, the total amount of lipids in sound wheat flours is low. Besides, the hydrolysis degree by the lipase action in dough systems is limited. Hence, the amount of mono- and diglycerides produced appears not to be sufficient to explain the effect of the lipase properties on breadmaking (175).

Although the expression of lipases of different origins in laboratory strains of Saccharomyces cerevisiae is well documented (177-179), the construction of industrial baker's yeast expressing lipases is now emerging. Monfort et al. (180) reported obtaining of lipolytic baker's yeast able to secrete active lipase by transformation with plasmids containing the LIP2 gene from Geotrichum sp. Recombinant lipase-2 protein exhibited biochemical properties similar to those of the natural enzyme (178). Fermented dough prepared with the recombinant strain rendered bread with higher loaf volume and a more uniform crumb structure than that prepared with control yeast. These effects were strengthened by the addition in the bread dough formulas of a preferment enriched in recombinant lipase 2 (180). Following the same approach, Sánchez et al. (181), described the expression of the lipA gene from Bacillus subtillis in baker's yeast. Unfortunately, the recombinant strain was unable to target the secretion of the heterologous enzyme into the culture media, even when a signal peptide was fused in frame to lip A.

11.4.4 Phytases

The low absorption of iron and zinc from cereal based meals has been ascribed to the high content of phytate (myo-inositol hexaphosphate), which forms insoluble complexes with these minerals at physiological pH values (182). Wheat grain contains endogenous 6-phytase (183), a phosphomonoesterase capable of hydrolyzing phytate to free inorganic phosphate and inositol via mono- to pentaphosphates. This phytase is activated during fermentation but not to such an extent that mineral bioavailability is greatly improved. It has been described as a 3-phytase activity characteristic of microorganisms (184), including baker's yeast (185). To reduce the phytate content of the whole wheat bread to a level considered not to affect mineral absorption, addition of phytase has been proposed (186). Furthermore, fungal phytase increases the specific bread volume and improves crumb texture and bread density (187). This improvement in bread making performance might be associated with the activation of endogenous a-amylase, due to the release of calcium ions from calcium-phytate complexes promoted by phytase activity.

As an alternative to the exogenous addition, phytase (phyA) from the fungus Aspergillus niger has been biotechnologically expressed in various hosts (188), and a S. cerevisiae strain producing an active extracellular phytase is available (189). The baker's yeast secreted phytase was found to be more thermostable than the commercial one, probably due to its high level of glycosylation. According to this, future improvements in commercial baking could be expected from the use of phytase-producing yeasts.

11.4.5 Other Enzymes

Enzyme technology is a major research topic in bread making. The utilization of new enzymes or applications is attracting increasing interest with the possibilities of generating improved products and processes or replacement of chemical substances (emulsifiers and oxidizing agents). In this way, transglutaminases, glucose oxidases, and laccases are a group of interesting enzymes for the baking industry (171, 190-192), because of their ability to oxidize or modify gluten proteins improving dough strength. Transglutaminase is also able to replace emulsifiers (191). This enzyme catalyzes an acyl transfer reaction between lysine and glutamine, stabilizing the gluten structure.

There is a growing interest in the use of some traditional enzymes, like proteases and lipoxygenases (170), because of the possibility of novel applications. For example, the controlled use of proteases with high specificity could increase the level of amino acids and peptides with properties as potential oxidants, taste enhancers, sweeteners, or bitter agents (193). In this scenario, the construction of recombinant strains for new or improved enzymes has started to render promising results. Thus, different pea seed lipoxygenases genes have been characterized and expressed in S. cerevisiae (194). Several examples of S. cerevisiae producing transglutaminases from different sources have been reported (195). Laccase-encoding cDNA has been isolated from the fungus Trametes versicolor and expressed in wild-type S. cerevisiae (196) or in recombinant strains overexpressing the protein Sso2p (117), a membrane protein involved in the secretion machinery. The glucose oxidase from Aspergillus niger has been also produced in S. cerevisiae strains (197).

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