5. Controles
5.4. Controles relacionadas con la disponibilidad
Although the use of industrial enzymes for starch hydrolysis did not become widespread until the 1960s, Payen and Persoz reported the reactions involved some 130 years earlier (Payen and Persoz, 1833). The enzyme used for the initial
dextrinization or liquefaction of starch is α-
amylase. The more scientifically correct or
systematic name for this type of enzyme is 1,4- α-D-glucan glucanohydrolase and it is also described by the following Enzyme Commission (EC) number, as laid down by the International Union of Biochemistry (IUB): 3.2.1.1. This number denotes that it is a hydrolase (3) of the glycosidase subcategory (3.2), which hydrolyzes O- glycosyl linkages (3.2.1). Because it is the first enzyme appearing in this category, its complete IUB number is 3.2.1.1. When faced
is effected by a class of amylases called
glucoamylases. Also included in Figure 6 is a
depiction of the mode of action of pullulanases
(α-dextrin 6-glucanohydrolase; EC No.
3.2.1.41), which belong to a category of starch- degrading enzymes known as debranching enzymes. The action of pullulanases concerns
the hydrolysis of 1,6-α-D-glucosidic linkages
in amylopectin, glycogen and their nascent limit
dextrins, generated by α-amylase activity. The
application of dedicated pullulanases in the ethanol industry is not widely encountered, although commercial preparations are available. This is due both to economic considerations and the fact that, in addition to a predominantly exo-
acting α(1→4) hydrolytic activity, glucoamylases
(see below) can also hydrolyze 1→6 bonds to a
limited degree. Given that the use of a glucoamylase component is absolutely required for efficient saccharification to occur, glucoamylase inclusion is normally relied upon to perform the dual functions of an amyloglucosidase and a debranching enzyme. Nevertheless, pullulanases deserve to be mentioned as a category of enzymes in their own right because they are likely to become much more affordable in the coming years.
In the beverage alcohol industry, ß-amylase
(1,4-α-D-glucan maltohydrolase; 3.2.1.2) is
another enzyme encountered in the starch
conversion process. Like α-amylase, this
enzyme cleaves α(1→4) linkages but attacks
starch in an ‘exo’ rather than an ‘endo’ fashion. The enzyme cleaves maltose (a disaccharide of glucose) in a stepwise manner from the non- reducing end of the starch polymer. The enzyme
cannot bypass α(1→6) branch points to attack
linear 1→4 bonds on the other side and
generates ß-limit dextrins as a result. Hence, the enzyme is most effective when used in conjunction with a debranching enzyme. ß- amylases are also utilized in the syrup industry for the production of high-maltose syrups from starch (McCleary, 1986). This enzyme represents a good example of the confusion that sometimes arises in enzyme terminology. One could easily be forgiven for questioning how a
ß-amylase can act upon α(1→4) linkages. The
main reason for this confusion is due to the fact that enzyme nomenclature is based upon the configuration of the released product rather than that of the bond being hydrolyzed. As ß-amylase with the IUB-designated nomenclature for
enzymes, the value of even a modicum of knowledge in the area of carbohydrate linkage terminology becomes apparent. For example, in
the case of α-amylase, one can tell from its
systematic name that it hydrolyzes 1→4 bonds
linking α-D-glucose residues. A further
characteristic of α-amylases is that they are
endo-acting enzymes, meaning that they attack the starch polymer from within the chain of linked glucose residues rather than from the ends. They
randomly cleave internal α(1→4) bonds to yield
shorter, water-soluble, oligosaccharide chains called dextrins, which are also liberated in the α-configuration. α-Amylases are metallo- enzymes and require calcium ions to be present
for maximum activity and stability. Finally, α-
amylases cannot cleave α(1→6) bonds and
bypass the branch points in amylopectin. When
this occurs, the residual products are called α-
limit dextrins.
Commercial sources of α-amylase are
produced mainly by Bacillus species, for example, Bacillus amyloliquefaciens and B.
licheniformis. Because cooking conditions for
starch will be dealt with in detail elsewhere, it is
sufficient to point out here that the choice of α-
amylase is based principally on tolerance to high temperatures and that this varies quite widely among enzyme sources. One common approach
is to cook the starch at approximately 105oC in
the presence of a thermostable α-amylase,
followed by a continued liquefaction stage at
90-95oC. The maximum extent of hydrolysis,
or dextrose equivalence (DE), obtainable using
bacterial α-amylases is around 40, but care
should be exercised not to overdose or prolong treatment since this can lead to the formation of
maltulose (4-α-D-glucopyranosyl-D-fructose),
which is resistant to hydrolysis by α-amylases
and glucoamylases. It must be remembered that the principal function of liquefaction is to reduce the viscosity of the gelatinized starch, thereby rendering it more manageable for subsequent processing.
The general action of amylases on starch is shown in Figure 6, where the first phase of enzymatic starch degradation, i.e. the production
of both dextrins and α-limit dextrins from starch,
is schematically depicted. As stated earlier, the next stage is termed the true saccharification phase, which under most practical circumstances
30 R.F. Power
catalyzes the removal of maltose units from starch, it produces what is known as a Walden inversion of the OH group at C-1, giving rise to
ß-maltose (Belitz and Grosch, 1999). Thus, the
products of bacterial and fungal α-amylases are
all in the α-configuration, while the products of
ß-amylases exist in the ß-configuration.
As mentioned, the true saccharification or conversion of dextrins to glucose is conducted, in the vast majority of cases, by glucoamylase,
although other enzymes, including some α-
amylases, have significant saccharifying activity in their own right. Glucoamylase is also known
as amyloglucosidase (glucan 1,4 –α-glucosidase;
EC No. 3.2.1.3) and its main activity is the
hydrolysis of terminal 1,4-linked α-D-glucose
residues successively from the non-reducing
ends of dextrins causing the release of ß-D- glucose. In other words, it is an exo-acting enzyme that cleaves single molecules of glucose in a stepwise manner from one end of the starch molecule or dextrin. Reference has also been made to the fact that glucoamylases can
hydrolyze α(1→6) bonds to a certain extent.
However, these branches are cleaved at a rate approximately 20 to 30 times slower than the
cleavage of α(1→4) bonds by the enzyme. Extra
glucoamylase can be added to compensate for this slower rate but this can cause undesirable side reactions to take place, whereby glucose molecules repolymerise in a reaction termed reversion, forming isomaltose and causing a decrease in glucose yield.
Glucoamylases are isolated from fungal
Figure 6. Schematic representation of the action of amylases on starch.
LIQUEFACTION
Production of dextrins andα-limit dextrins byα-amylase AMYLOSE AMYLOPECTIN DEXTRINS α-LIMIT DEXTRINS SACCHARIFICATION Hydrolysis of dextrins to fermentable sugars GELATINIZATION
Heat and moisture solubilize starch
α-amylase
endoenzyme
hydrolyzes random α(1-4) bonds
α-amylase α-amylase α-amylase Pullulanase (debranching) Glucoamylase α(1-4), α(1-6 ) bonds Glucoamylase STARCH amylose + amylopectin Slurry Cooking
FERMENTABLE SUGARS Glucoamylase
exoenzyme
hydrolyzesα(1-4) bonds from non-reducing ends hydrolyzesα(1-6) bonds more slowly
sources such as Aspergillus niger and Rhizopus
sp. Fungal enzymes, by nature, are less
thermotolerant than their bacterial counterparts and therefore the temperature maxima for
glucoamylases tend to be in the region of 60oC
while their pH optima normally lie between pH 4.0 and 4.5. These conditions of temperature and pH can cause significant problems in plants using dedicated saccharification tanks because they are quite conducive to the growth of certain bacterial contaminants. The claimed benefit of pre-saccharification of mash prior to fermentation is that a high level of glucose is made available at the start of fermentation. However, in addition to infection problems, such an approach can lead to the generation of non- fermentable reversion products, such as isomaltose.
Because of the drawbacks of mash pre- saccharification, a popular approach is to saccharify simultaneously with fermentation (SSF or Simultaneous Saccharification and Fermentation). The requirement for high glucose concentrations at the start of fermentation is met by the addition of glucoamylase during the filling of the fermentor. Although the concern with such a system is that glucose may become limiting during the fermentation, this is not a problem in practice and is merely a case of metering in the correct amount of enzyme. Glucose is used up as it is produced and thus there is little risk of reversion reactions occurring. An additional attraction to this approach is that it lends itself to the use of somewhat non- conventional enzymes, which can boost ethanol
yield by liberating fermentable sugars from non- starch polysaccharide sources. For example, multi-enzyme complexes isolated from fungal cultures grown on fiber-rich, semi-solid or surface culture systems, contain a mixture of amylolytic, cellulolytic and other polysaccharidase activities which have temperature and pH optima that quite closely match those found in distillery fermentors. One commercial preparation, Rhizozyme™, is a surface culture enzyme gaining increasing use in the fuel ethanol industry. These enzymes satisfy the requirement to saccharify dextrins to glucose but have the additional activities required to attack, for example, ß-linked glucose polymers, if present.
Summary
The objective of industrial alcohol production is to produce the highest ethanol yield possible. This requires the greatest attainable conversion of starch to fermentable sugars because yeast cannot use starch in its native state. The use of starch-degrading enzymes represented one of the first large-scale industrial applications of microbial enzymes. Traditionally, two main enzymes catalyze the conversion of starch to glucose. In the first stage of starch conversion, α-amylase randomly cleaves the large α-1,4- linked glucose polymers into shorter oligosaccharides at a high reaction temperature. This phase is called liquefaction and is carried out by bacterial enzymes. In the next stage,
Table 3. Enzymes used in the starch hydrolysis process.
Enzyme EC number Source Action
α-amylase 3.2.1.1 Bacillus sp. Only α-1,4-linkages are cleaved to produce α-dextrins, maltose and oligosaccharides G(3) or higher
Aspergillus oryzae, A. niger Onlyα-1,4-linkages are cleaved producing α-dextrins, maltose and G(3) oligosaccharides
α-amylase 3.2.1.1 Bacillus amylosacchariticus Cleavesα-1,4-linkages yielding α-dextrins, maltose, G(3), G(4)
(saccharifying) and up to 50% (w/w) glucose.
ß-amylase 3.2.1.2 Malted barley α-1,4-linkages cleaved, from non-reducing ends, to yield limit dextrins and ß-maltose.
Glucoamylase 3.2.1.3 Aspergillus sp. Rhizopus sp. α-1,4 and α-1,6-linkages are cleaved, from the non-reducing ends,
to yield ß-glucose.
32 R.F. Power
called saccharification, glucoamylase hydrolyzes the oligosaccharides or dextrins into free glucose subunits. Fungal enzymes are normally employed to effect saccharification and these operate at a lower pH and temperature
range than α-amylase. Depending on the
industry and process involved, one sometimes encounters additional enzymes, such as pullulanase, which is added as a debranching enzyme to improve the glucose yield.
Despite the intricate nature of enzyme nomenclature and the rather strict reaction conditions which must be adhered to in order to achieve maximal enzyme performance, their purpose is very simple: to provide fuel to yeast for the biosynthesis of ethanol. This goal must not be lost sight of and new approaches, enzymatic or otherwise, need to be explored in an effort to liberate additional fermentable sugars from common cereal sources. The application of surface culture and fungi that produce multi-enzyme complexes, encompassing several polysaccharidase activities in addition to the requisite, traditional, amylolytic activities, represents one promising approach towards maximizing the release of utilizable carbohydrate from both starch- and non-starch- based cereal polysaccharides.
References
Belitz, H.-D. and W. Grosch. 1999. Food
Chemistry. (M.M. Burghagen, D. Hadziyev, P.
Hessel, S. Jordan and C. Sprinz, eds). Springer- Verlag, Berlin Heidelberg. pp. 237-318. Kelsall, D.R. and T.P. Lyons. 1999. Grain dry
milling and cooking for alcohol production: designing for 23% ethanol and maximum
yield. In: The Alcohol Textbook, 3rd ed.(K.A.
Jacques, T.P. Lyons and D.R. Kelsall, eds). Nottingham University Press, UK. pp. 7-24. Lehninger, A.L. 1982. Principles of
Biochemistry. (S. Anderson and J. Fox, eds).
Worth Publishers Inc. New York. pp. 277-298. McAloon, A., Taylor, F., Yee, W. Ibsen, K. and R. Wooley. 2000. Determining the cost of producing ethanol from corn starch and lignocellulosic feedstocks: A Joint Study Sponsored by U.S. Department of Agriculture and U.S. Department of Energy. National Renewable Energy Laboratory Technical Report/ TP-580-28893.
McCleary, B.V. 1986. Enzymatic modification of plant polysaccharides. Int. J. Biol. Macromol. 8:349-354.
Payen, A. and J.F. Persoz. 1833. Mémoire sur la diastase, les principaux produits de ses réactions, et leurs applications aux arts industriels. Ann. Chim. Phys. 53: 73-92.