http://hubris.engin.umich.edu:8080/Beer/Threads/Threads/thread.973554365.html
I think a little enzyme note is in order.
Enzymes are proteins, and are created in a nearly direct
transcription process from DNA to RNA to protein. Enzymes and their
remarkable catalytic activity are a primary expression of genes.
These long strings of amino acids are have extremely complex shape or
conformation. They form spirals like DNA and amylose, but also fold
and turn sharply. They forms weak molecular bonds between the folds.
The various amino acids have distinct electrical properties and so
along the length of the protein the electrical potential varies. The
internal electrical attractions also determines the shape of these
ribbons of amino acid.
Enzymes just speed up reactions, they are not used up, nor do they
supply energy. They just make reactions which are already happening
very slowly, suddenly happen much faster. Imagine a billiard table
without pockets (where would the physical sciences be without
billiard analogies?). The lowest energy state for the ball is on the
floor, while the table is a higher potential energy state. The balls
usually stay on the table during normal play, because they don't have
the energy to make it over the bumper. If you supply a lot of kinetic
energy then once in a long while a ball goes over the bumper and hits
the floor. Adding enzymes is like lowering the bumper height. No
energy is added to the balls, but the rate of change from high to low
energy state is vastly increased.
Hydrolytic enzymes [amylases, glucanases proteases and peptidases in
malt] require substrate (starch, protein etc) AND WATER. Water is a
key reactant in the breakdown process. The hydrolysis reaction breaks
a chemical bond in starch, for example, and one side of the broken
bond gets a hydrogen (H) from water, while the other side gets the
hydroxyl group (OH). So busting up all the bonds in your malt
actually uses up a little water.
Next we need to consider the kinetic energy amongst the molecules.
The amount of energy needed to tear apart a protein or starch
molecule is extremely high - like having billiard table bumpers a
foot high. When enzymes are added the energies required drop by a
factor of 2 to 10, but they are still much higher than the typical
energy of a molecule at room temp. For example a particular
peroxidase enzyme lowers the 'bumper' energy from 76kJ/mol to
30kJ/mol. But at room temperature the average molecule has only about
3-4kJ/mol, and raising the temperature by 10C increases the average
kinetic energy by less than 2%.
Fortunately the energy is not evenly distributed between the
molecules. A small, but appreciable number of molecules carry the
requisite 30kJ/mol for the reaction above, and many orders of
magnitude fewer carry the 76kJ/m needed for the non-enzymatic
reaction. Also the number of these very high energy molecules
increases almost exponentially with temperature, leading to the
familiar rule of thumb that many reaction rates double per 10C temp
increase. There are other factors which make this rule imprecise, but
the main points are that the number of reactions with sufficient
energy is vastly increased (often by a factor of 10^6 to 10^10) by
the presence of enzymes. Also that this number roughly doubles per
10C increase in our domain of interest (20C to 100C).
If you bring together water, substrate and enzyme - all in the right
orientation and conformation, with sufficient kinetic energy, the
hydrolysis breakdown occurs. But at what rate ?
TEMPERATURE:
As we have seen, temperature increases cause an almost exponential
increase in the number of collision in which the reaction might
happen. So temperature is a major factor. Instead of increasing at 2X
per 10C as a simple analysis would suggest, enzyme reaction rates
typically increase from
1.2X up to 3X per 10C.
CONCENTRATION:
The reactant concentrations impact the reaction rate. Given a
solution of amylase enzyme, an increasing amylose concentration at
first causes a near linear increase in reaction rate, but eventually
when enough amylose substrate is added the enzyme is effectively
'saturated' and can react no more quickly. The curve this represents
and the equations that fall out of this observation are attributed to
Michaelis and Menten of almost a century past. (see crude graph below)
REACTION RATE
|
| _____----------------- - Vmax
| . -
| `
| /
| -
| /
| /
|/_______________________ reactant concentration
Water is a reactant in our mashes too, but since it is also our
solvent the matter is a bit confusing to think about. For a *fixed*
concentration of amylase and amylose, increasing the water
concentration results in a curve similar to the one above. However if
we just add water without holding the enzyme and substrate
concentrations fixed, then we just end up diluting these. In other
words a pound of malt thrown in Lake Michigan takes forever to
convert because the enzyme and starch concentrations are so low and
not directly because the water concentration is high.
That is really about it - except of course for the fine print ..
FINE PRINT:
1/ The instantaneous concentration of enzyme changes. Enzymes go into
solution during the mash-in, and in solution their likelihood of
interacting is higher. The more interesting factor is the denaturing
of enzymes. When enzymes break their internal weak fold to fold bonds
they lose their shape and so their effectiveness. In a few cases this
denaturing is reversible - but this is rare. The major cause of
denaturing in the mash is temperature. If enzymes are involved in
collisions (or vibrations) too energetic then their internal
structures change. They are still proteins, but they are no longer
enzymes. Under a fixed set of conditions, the rate at which enzyme
denature due to temperature is akin to the way radioactive material
decays, or the charge on a capacitor bleeds off. It makes sense to
describe it in terms of a half-life. A 1.25qt/lb 65C mash (and other
conditions of pH etc), the half-life of beta-amylase was about 16
minutes. This means that the first 16 minute mash interval will have
twice the activity of the second interval and four times the activity
of the 3rd interval, and so on, assuming other conditions are
constant (which they are usually not). The enzyme decay rate with
increasing temperature is also exponential in nature, but is much
greater than the activity increase and usually in the range of 6X to
36X per 10C increase !! This means the half-life time decreases
markedly with temperature increase.
If you 'do the math' you will find that there is an optimal
temperature for a mash enzyme ONLY if you specify the time duration
of the mash. For a 15 minute mash, maybe 70C is optimal for beta
amylase (!!), while for a 2 hr mash perhaps 58C is optimal.
2/ The amylopectin in a mash ties up an incredible amount of the
water as gel and so this water is unavailable for the enzymatic
hydrolysis. Amounts of water much less that 1.1 qt/lb of malt cause a
tremendous drop of in reaction rate. This is similar to the 'knee
point' in the crude graph above. Of course the gel changes throughout
the course of a real mash so this factor is dynamic.
3/ The enzyme activity varies with conditions. The enzyme, as
previously stated, has a precise conformation that is effected by the
electrical charges that it carries. These electrical attractions
cause minor changes in the enzyme shape which in turn may have a big
impact on the enzyme activity. Free salt ions or a change in pH can
have a profound effect on the enzymes charges, shape and activity.
Contrary to some previous posts, these changes are NOT typically
denaturing. They usually effect activity, but they do not destroy the
enzyme.
4/ Some enzymes also have cofactors, additional molecular 'partners'
which either allow or improve the enzyme catalysis. Each
alpha-amylase molecule for example requires a calcium ion for it's
activity. It is thought the Ca ions charge effects the enzyme
conformation. Many vitamins and minerals are enzyme co-factors.
5/ Substrate stabilization. Increasing the amount of substrate and
decreasing the amount of water often has the effect of making the
hydrolytic enzyme more stable. In the extreme case very dry but
starch rich malt, is kilned at 120C and above - yet the enzymes
survive for hours. In less extreme cases thick mashes *may*
demonstrate greater proteolytic or beta-amylase activity than
expected in a thin mash at the same temperature, *BUT* the low free
water concentration also limits the reaction rate. I am aware of this
method being used, for example to get a protein rest from infusion
hardware, but generally I think it is of limited value and difficult
to control accurately as compared to time and temperature control in
a step mash.
6/ Product inhibition. Some enzymes, like beta-amylase can have their
activity inhibited by the presence of their own product (maltose).
Sometimes the product can also stabilize as well as inhibit the
enzyme. Perhaps in a very high gravity low temp mash this has an
impact but I doubt that it is usually a significant factor compared
the temperature denaturing loss of beta-amylase
7/ Other stuff. There are a myriad of other effects that are small
but may add up to something. Pumps and stirrers can denature enzymes
through shear force damage. Chemical reactions may change the enzyme,
proteases and peptidases may destroy some enzymes (which are
proteins). Extreme pH conditions may denature. Of course combinations
of the above may have a synergistic effect. The 'wrong' pH may make
enzymes more susceptible to shear damage of thermal denaturing.
=== dogma challenged ...
As a practical matter, the enzyme to starch & protein ratio's are
fixed by our choice of mash bill. I personally think that water:grist
ratios below 1.1 qt/lb are questionable, and that figures around
1/5qt/lb are probably near optimal. Studies show small but real
increases in extraction and enzyme activity to 2qt/lb and even
beyond, but I would reserve these thinner mashing techniques to
cereal mashes where the undegraded amylopectins require vastly more
water.
Any mashing with brewers malt must take into account the fact that
not only is alpha-amylase less temperature sensitive than
beta-amylase, but it exists (in terms of activity) in vastly greater
quantities. (~20X) Because of this, saccharification rests in mashing
should be considered an exercise in getting just the desired amount
of beta-amylase activity. Sufficient alpha-amylase exists so that a
single infusion rest at 80C(176F) gives completely normal extract
levels, and sufficiently low starch levels to be considered a
complete conversion !! [only at 85C do starch levels rise
dramatically].