Enzymes

Heres a useful email from Stephen Alexander about enzymes, explaining how they affect the rate of a reaction, and how they work in the conversion of starch to glucose.


From The Homebrew Digest
Date: Fri, 30 Jul 1999 15:37:24 -0400
From: "Stephen Alexander"
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 1.1qt/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].


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