Why does amylase work at high temperatures?

Alpha amylase is optimized to work in germinating barley seeds. They certainly don't get up to the temperatures we use to mash. Why is the optimum temperature, then, for mashing 150-160F? That seems to be WAY outside the logical range for this enzyme.

There are many enzymes, and it's oversimplistic to say that alpha amylase only "works" at XXX temperature. Here's some good but simple reading on enzymatic activity and mash temps, along with a chart: http://www.howtobrew.com/section3/chapter14-1.html

apeltes said:

Alpha amylase is optimized to work in germinating barley seeds. They certainly don't get up to the temperatures we use to mash. Why is the optimum temperature, then, for mashing 150-160F? That seems to be WAY outside the logical range for this enzyme.

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Lots of organisms (plant, animal, bacteria) use alpha amylase so it is wrong to say that it is adapted to work in germinating barley.

Because evolution does not optimize. Alpha enzyme is most efficient (aka fastest) at 160F, but it works fine, if somewhat slower, at room temperature.

Survival of the "good enough", not the fittest, is the truth. Just look at what a lousy arrangement the human eye is, but it's good enough.

apeltes said:

Alpha amylase is optimized to work in germinating barley seeds. They certainly don't get up to the temperatures we use to mash. Why is the optimum temperature, then, for mashing 150-160F? That seems to be WAY outside the logical range for this enzyme.

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The ~150F mash temperature is the best for brewing beer because that provides the maximum alpha amylase activity for breaking down starch chains into fermentable sugars. As has been said above that is not the only thing alpha amylase might do nor does it mean that alpha amylase does not work at all at different temperature ranges.

david_42 said:

Because evolution does not optimize. Alpha enzyme is most efficient (aka fastest) at 160F, but it works fine, if somewhat slower, at room temperature.

Survival of the "good enough", not the fittest, is the truth. Just look at what a lousy arrangement the human eye is, but it's good enough.

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I'd say it is perfectly optimized to do what it needs to at lower temperatures. The barley produces amylase after it germinates (malting), to turn stored starches into food. It has to be slow for a few reasons.
A growing plant doesn't need a ton of sugar all at once. but rather a slow continuous supply
If you convert the starch into sugar all at once, the water activity inside the seed would drop like a rock and suck the moisture from the growing sprout.

The alpha amylase in barley evolved to work best in germinating barley seeds (and evolution, natural selection specifically, DOES optimize things).

Any enzyme tends to have one temperature at which it works best. Any higher or lower, and it loses efficiency. For example, human digestive enzymes work best at approximately 98F. Any higher or lower, and they become less effective.

Why does mashing work best at almost 170F? Why doesn't mashing work best at the temperature at which barley germinates?

I'm way out of my league here but will venture a WAG. The efficacy of an enzyme depends upon its shape and its shape is controlled by the charges on the acid residues. Whether an acid residue gives up its proton or not depends on pH and the pK for that residue and the pK depends in turn on temperature. Thus temperature effects the conformation of the enzyme. Coupled with that is the fact that reactions generally take place faster at higher temperatures. OTOH, if the temperature gets too high the enzyme is denatured. Put all these together (if I'm right in my hypothesis) and there is an optimal temperature and pH for each enzyme whether this be the temperature at which it operates in nature or not. Amylases do not need to work at optimum rate to perform their functions in nature nor indeed in brewing. But in brewing speed is important. We don't want to have to wait overnight for the starches in our mashes to convert.

(and evolution, natural selection specifically, DOES optimize things).

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No, check out the efficiency of photosynthesis using chlorophyll. The most primitive photovoltaic cells do a better job.

Evolution via natural selection doesn't necessarily (or usually) produce the best possible configuration, but that's not what I mean by "optimize". I'm saying that natural selection favors the most adaptive version of a trait given a range of available genetic variation. Thus, for enzymes, we can usually trust them to work best under the conditions in which they evolved.

I think the best hypothesis we have so far is that alpha amylase obviously works well within a very broad temperature range. It's not as "sensitive" as the enzymes I'm more familiar with. For example, many mammalian digestive enzymes slow significantly at a few degrees above normal body temperature (or below). Perhaps we need to mash at such high temperatures to compensate for the reduction in concentration produced when we crush and mix with water? Within limits, reduced concentration equals reduced reaction rate.

I believe your issue comes in the misconception of what optimized for brewing and biologically optimized.

Biologically Alpha Amylase has a HUGE range of stable temperature operation. It is active from basically 0C up to 90C, which makes it ideal in starch conversion in a wide range of organisms (including humans).

Now with brewing your balancing what enzymes are in your mash, their working temps, and what you want to do. For example while your mashing at say 130F you'll have a lot of things going on, alpha and beta amylase and upward range of the proteases. The proteases are actively breaking down all the proteins in the mash INCLUDING alpha and beta amylase so obviously this is not a good place to be for too long.

So Alpha Amylase is very optimized (optimized DOES NOT MEAN MINIMIZED) for what it does and is widely used, and that we should all be thankful for. I hope this helps

I agree that alpha amylase can certainly do its job at nearly any temperature. It's a very forgiving enzyme. But any enzyme has, within the range of temperatures within which it will work, a window within which it works best (fastest)... depending on temperature and pH. For human amylase, I'm willing to bet it works best at around 98F. For barley, it should be closer to the temperature it usually experiences during germination, which surely isn't 160F (where we mash). The only thing, then, that makes sense is that barley's amylase isn't working as fast as it can when it does it's job during germination. If that were the case, we would mash at around 70F or 80F, I guess.

apeltes said:

But any enzyme has, within the range of temperatures within which it will work, a window within which it works best (fastest)... depending on temperature and pH.

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I completely agree. That window is around (actually a little bit higher then) room temp. The point I was trying to make is that optimized for brewing, the 154-156 range, is that way because of lack of other enzyme activity not the speed of which alpha amylase works.

apeltes said:

For human amylase, I'm willing to bet it works best at around 98F. For barley, it should be closer to the temperature it usually experiences during germination.

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Correct, but it doesn't mean it can't work at higher temps effectively.

apeltes said:

The only thing, then, that makes sense is that barley's amylase isn't working as fast as it can when it does it's job during germination. If that were the case, we would mash at around 70F or 80F, I guess.

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Your thinking of this backwards. It works slower at mash temps, but it still works fine and decently fast (remember an hour is a LONG time in enzyme world). The reason we mash high as apposed to room temp is to denature enzymes we don't want working and just lucky enough that alpha and beta amylase can withstand the temps. If it wasn't the case we would have to boil the extract then add our mash enzymes then boil it again (with hops). I hope I made myself a little more clear.

It is a happy coincidence for us that it is this stable. The half lives of proteins varies immensely. Some are stable for only minutes in their native environment, others are stable for a very long time - like the chitin and the protein in shells. It depends on the structure of the protein.

Once could conduct a mash at 50 F. It would take several days to convert, and during that time, a host of microbes present on the grain would start chewing up the released sugars. This is actually probably what happened on the very first beers (probably more like 90 F though).

Germination is a HIGHLY coordinated process. The seedling will only break down starch to make sugars as fast as they can be utilized for growth. Lots of excess sugar around is an open invitation for hungry microbes. These seeds are living in the soil which is loaded with organisms that would love to get at the sugar for themselves.

As an example, super sweet corn seeds are a pain to germinate in the field. The seeds don't store starch, just simple sugars. One result is the seeds really shrink and shrivel - and crack. Put them unprotected in the soil and the microbe party light goes on. Very few seedlings will survive without a seed fungicide treatment.

And, germination does in fact occur more rapidly in a warmer than room temp environment. Lots of pro's in Nurseries utilize heat mats for seed starting to increase yield.

My confusion is probably based on the chart below (from: http://www.realbeer.com/jjpalmer/ch14.html ).

It shows each enzyme having a "range", so I assumed alpha amylase works poorly below 146F. Is that not the case?

apeltes said:

My confusion is probably based on the chart below (from: http://www.realbeer.com/jjpalmer/ch14.html ).

It shows each enzyme having a "range", so I assumed alpha amylase works poorly below 146F. Is that not the case?

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These ranges are specific to beer production. Plain and simple.

For temp optima related to germination, look at malting processes. Floor temps etc... Those are most definitely NOT kept at 150*F+ but are monitored for optimal success.

Again, the range is for practical brewing application. We can't take 3 days to mash at 90F for a couple reasons. First, you'd get lacto infection which is great if you like Berliner Weisse but... Second, you're under the gelatinization temp of barley so the yield would suck badly. The reason why the brewing optimum is in the range is based on both the gelatinization temp and the speed at which you get full starch conversion.

Germination is a HIGHLY coordinated process. The seedling will only break down starch to make sugars as fast as they can be utilized for growth. Lots of excess sugar around is an open invitation for hungry microbes. These seeds are living in the soil which is loaded with organisms that would love to get at the sugar for themselves.

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Thumbs up on that. Plants have to adapt to biological and nonbiological stresses/threats by changes in gene expression only, they can't run away.

I think the consensus is that barley amylase is well-adapted to operate at germination temperatures. Although it works at mash temperatures, it doesn't work as well... but that's fine. We want to convert at the top of the temperature range to suppress other enzymes and to prevent infection.
Fair summary?

Question: What would happen if malt were mashed at 80F? Would the amylase succeed in quickly converting the starch? Would something interfere, since mashing does not happen in a living seed/embryo? Is the enzyme too dilute once mixed with so much water?

Within the range at which an enzyme is active, it's activity generally doubles for each 10degreesC increase in temperature. The reason for the boxes around alpha and beta amylase in your chart are to indicate the relative activities. The beta enzyme starts to denature around 155 or so, so the alpha (which is slightly more stable) dominates the profile from that point, until it denatures.

These enzymes hang around in seeds for a very long time. And enzymes that are stable over time like that also tend to be more stable to temperature. Which is why it remains active at what could be considered outside its normal range.

apeltes said:

I think the consensus is that barley amylase is well-adapted to operate at germination temperatures. Although it works at mash temperatures, it doesn't work as well... but that's fine. We want to convert at the top of the temperature range to suppress other enzymes and to prevent infection.
Fair summary?

Question: What would happen if malt were mashed at 80F? Would the amylase succeed in quickly converting the starch? Would something interfere, since mashing does not happen in a living seed/embryo? Is the enzyme too dilute once mixed with so much water?

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Someone earlier mentioned it, but it may have been lost among the sea of people who didn't understand your question (which is an entirely reasonable one).

Enzymes usually run faster at higher temperatures, and barley's alpha amylase does too. They also fall apart faster at higher temperatures. That barley's alpha amylase (which, contrary to what countless people here have said, isn't identical to the alpha amylase in everything else) is stable at high temperatures is probably, in large part, just our good luck. It's not completely unexpected, though: lots of enzymes don't get denatured until fairly far outside the organisms standard temperature range. (We can also view this from the opposite point of view: if barley's alpha amylase wasn't stable at high temperatures, we'd be brewing with some other grain and asking the question about it.)

The second element of your question is why barley alpha amylase's peak efficiency isn't more closely aligned with the temperatures the organism would experience in nature. As was mentioned earlier, this is probably mostly because it is not advantageous for the plant to run its enzymes that quickly. Germination is a slow, coordinated process, in which starch is converted to sugar at a rate that is required by the organism. It does the barley seed no good to bury itself in a sea of sugar it is not yet ready to use -- it's probably a disadvantage, in fact, because of the increase in osmotic pressure and increased infection risk.

So, in summary: Why doesn't barley's alpha amylase run fast at the temperatures it experiences during germination? Because faster isn't better, in this case. Why, then, can we use it at higher temperatures at all? Probably mostly luck, but not a huge amount of it.

Probably mostly luck, but not a huge amount of it.

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So, it comes down to the beer wisdom of Ben Franklin: It works because God loves us and he wants us to have beer to be happy!

Seriously though, good summary, ni.

I see that this is an old thread (Over 10 years!) but this is an interesting question that I dont think has been addressed scientifically. I am a research scientist and deal with structure-function questions all the time. I have recently asked myself about the phenomenon of why amylases work at the temperatures in the mash where most plant proteins would become unfolded and non-functional.

Higher temperatures does not mean higher efficiency. Higher temps can increase efficiencies, but only within a small range. Outside that range, proteins/enzyme start to unfold (denature) when they get to hot. Evolution also does an excellent job of optimization, not perfect but excellent.

I previous post about the slower rate of enzyme function at germination temps makes sense as this would allow for slow release of sugars as the plant develops. To have all the amylose/amylopectin converted to simple sugars all at once would be detrimental and this as an evolutionary mechanism to limit or slow function is a reasonable hypothesis. pH is also a contributing factor but most biological systems function at a very narrow pH range (unless your bacteria!).

For my the question is why or how does this structure change with temperature to allow it to function differently. It may not matter in the end and while home brewing but for me it is still and interesting question.