Basic Brewing Enzymology

A beginner’s primer on enzymes in beer Presentation Summary

This seminar is a companion to the following presentation, Practical Enzymatic Brewing. This presentation is beginner level, covering: • Background biology and chemistry of enzymes, proteins, and carbohydrates; • Enzymes endogenous to malted grains (how and why they form); • The typical process of brewing, as viewed through the lens of enzyme activity (from mash-in to clarification). Alan B. Windhausen Head Brewer

Holidaily Brewing Learning Objectives

• Understand the foundation of enzymology (biology and chemistry of enzymes); • Review the biological and chemical nature of the components to beer; • Know the sources of endogenous enzymes in malt; • Understand the role of enzymes in a basic brewing process. Outline

Definitions and Background Endogenous Enzymes in Beer • What are Enzymes? • Malting • Background and basics • What are Proteins? • Germination and Modification • Kilning • What are Carbohydrates and Starches? • Mashing and Boiling • Enzymes, Functions, and Ranges • Endogenous vs. Exogenous • Adjuncts • Enzyme Naming Conventions • Fermentation • Dry Hop Creep Definitions and Background What Are Enzymes?

• They are proteins (usually) with a job.

• Specifically, made by a living organism to catalyze a biochemical reaction

• Not all enzymes are proteins, but the vast Glucosidase, breaking Maltose into two majority are. Glucose.

• Biological catalysts.

• Catalysts reduces the amount of energy needed for the reaction.

• Thus, it will occur faster and with greater frequency. A simplified graph of the breakdown of glucose. • Catalysts are not consumed in the reaction! © Mcy Jerry, 2005, Wikimedia Commons What Are Enzymes? (cont’d)

The molecules that Sometimes, more Enzymes are enzymes act upon are than one substrate structured to only called “substrates.” is involved. interact with a very specific substrate (or substrates) “substrate: a substance acted upon (as by an enzyme)” – Merriam Webster

Two substrates interacting with an Lock and Key model of enzyme enzyme bonding From https://openstax.org/books/anatomy-and-physiology/pages/2-5-organic- compounds-essential-to-human-functioning © Saylor Academy, 2012 What Are Proteins? • Proteins are long-chain molecules comprised of amino acids.

• 4 levels to their structure: • Primary – the sequence of the amino acids.

• Secondary and Tertiary – the ‘folding’ and ‘pinning’ of the molecule into a specific shape.

• Quaternary – multiple separate chains forming a single Structure of Proteins structure. Schmid, Silvan. (2020). Immobilization of DNA and protein to polymerized SU-8 photoresist investigated with microarray assays. What are Proteins? (cont’d)

Proteins only function when the entire structure is intact! Factors that will break down that structure: • pH’s outside their ‘functional’ range. • High temperatures. Relatively low temperatures are fine, but reactions slow down in cold. A protein broken down to just primary structure is fully ‘denatured’. Denatured proteins no longer function as intended. Denatured enzymes will not perform their task. What are Proteins? (cont’d)

A graph of enzyme activity vs temp. © Thomas Shafee - Own work, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=47436421 Carbohydrates and Starches Carbohydrates: molecules built out of simple sugars.

Two sugars together are called disaccharides.

Long chains of sugars (polysaccharides) create larger carbohydrates.

Cellulose is made of β-glucose, while starches are made of α- glucose. Structure of mono- and di-saccharides © Christine Dobrowolski, CC BY-SA 2.0 Carbs and Starches (cont’d) Starches: long chain-like molecules made up of many glucose.

Amylose - Straight chains of sugars

Amylopectin - Branched

Enzymes will interact with only one of type, or even a specific section. Amylose and Amylopectin, acted upon by α-amylase The filled circles indicate glucose subunits with α-1,6, glycosidic linkages

Margaret E. Smith PhD DSc, Dion G. Morton MD DSc, in The Digestive System (Second Edition), 2010 Endogenous and Exogenous Endogenous Endogenous: “internally originating” (in) • Created inside an organism to perform a task critical to that organism’s survival.

Exogenous: “externally originating” • Added from an external source (i.e. by you) Endo- & Exogenous (cont’d) Commercial enzymes: • From bacteria or fungal sources, sometimes yeast – sometimes GMO. • Older sources: animal (α-amylases) or plant sources (papain).

Exogenous doesn’t always mean commercial: • Traditional Chicha is chewed to convert maize starches.

A commercial Chicha (Dos Luces) Courtesy of Dos Luces Brewing Enzyme Naming • Pre-1955, named for the substrate, product, discoverer, or damn-near randomly.

• Today, enzymes common names are “substrate + ase.” • E.g. lactase breaks down lactose.

• Sometimes, the action being performed is added: • E.g. β-glucan solubilases solubilize β-glucan.

Role of Solubilases in Cell Wall Degradation Courtesy of Canadian Malting Barley Technical Centre Enzyme Naming (cont’d) Enzyme Code (first numbers only): • The common or ‘trivial’ name for an EC 1 – oxidoreductases (perform oxidation-reduction enzyme is typically used. reactions on substrates) EC 2 – transferases (transfer molecular components between substrates) • The ‘proper’ enzyme name is further EC 3 – hydrolases (breaks bonds on a substrate broken down: using hydrolysis, the cleaving of bonds with an H+ • List all substrates and OH- from water – amylases are hydrolases) • State exact reaction EC 4 – lyases (break apart bonds on a substrate without using hydrolysis) • E.g., α-amylase is “4-α-D- glucan glucanohydrolase.” EC 5 – isomerases (isomerize substrates – that is, they rearrange the components of a substrate) EC 6 – ligases (join together substrates, while also • The Enzyme Code is a number used using a molecular energy source called ATP) for research EC 7 – translocases (added 2018 – transport ions or • α-amylase is 3.2.1.1 molecules across membranes in cells) Endogenous Enzymes in Beer Malting • The process of transforming grains (or seeds) into transportable packets of starches, enzymes, and proteins / amino acids.

• I.e., the breaking down cell walls and proteins while preserving the starch granules and enzymes.

• I.e., maltsters trick grain into freeing their starches, and brewers trick that into becoming yeast nutrients, because yeast has tricked us all into feeding it. Modification of barley, two paths Michael Lewis and Tom Young, Brewing (2nd edition), 2002) Malting (cont’d)

Malting consists of: Steeping: Germination: And Kilning:

Hydrating the grain Enzymes break down Halts modification, kick-starts the stored nutrients for the creates flavor. process. plant.

Allows chemicals to “Modification”: move freely. conversion to usability for the plant (and brewer). Germination

1 – The hydrated embryo eats sugars in its immediate vicinity.

2 – Once those are depleted, Gibberellins are activated.

3 – These hormones trigger the release or production of specific enzymes.

Enzyme production during malting Hans Sejr Olsen. Enzymes in brewing. Biokemisk Forening. 2008 Germination (cont’d) The types of enzymes produced are: Cellulose digesting And Starch Proteases: enzymes: digesting enzymes: (α- and β-amylase) – break Break down proteins, the Break down the walls that down starches into sugars. grain uses throughout the enclose the starch granules. process for various Brewers need these enzymes purposes. Principal enzymes are β- to be created but to not break glucanases (for β-glucans), down the starches yet. Creates Free Amino and pentosanases (such as Limit-dextrinase and other de- Nitrogen, critical for yeast xylanases). health. branching enzymes are also created.

Check out the CBC 2020 presentation on Malt COA’s! Germination (cont’d) Germination (cont’d)

Solubilization: - Xylanase - Acetyl xylan esterase - Feruloyl esterase - Arabinofuranosidase - Carboxypeptidase

Hydrolysis (breakdown) - Endo- and Exoglucanases - Glucosidases - Xylosidase Germination (cont’d)

Protein matrix breakdown: (Proteases) - Endopeptidases - Exopeptidases Germination (cont’d) Well-modified malt: • Cell walls and matrix broken down. • Starch and amylases intact for the brewer to control.

Cell Wall breakdown during modification Poorly modified malt Courtesy of Canadian Malting Barley Technical Centre • Has remaining cellular matrix. • Sticky long-chain molecules remain.

Barley modification by day Gianinetti, Theory in Biosciences, 2008 Kilning Suspends modification, leaves starches and amylases intact. • Allow enzymes to attach to remaining matrix (preserves structure). • High temperatures and longer times breaks down enzymes.

Caramel and crystal malts rehydrate ‘green’ malt prior to kilning / roasting. • Sugars form, then are caramelized

Munich 20L Malt vs Caramel 20L David Richter, Briess Malt and Ingredients – blogpost, Jan. 2018 Mashing Finally, the brewhouse!

Goal of Mashing: • Solubilize the soluble malt matter. • Gelatinize starches and convert into sugars. • Release and solubilize other desirable malt components. • All of this relies on the endogenous enzymes preserved from malting.

Triple mash process AEE Institute for Sustainable Technologies, technology wiki Mashing (cont’d) Enzyme Temperature Range Denatures pH Range Function

α-amylase 150-160 oF (66-71 oC) ~170 oF (77 oC) 5.3-5.7 Cuts larger starches (Ca2+ randomly stabilized) β-amylase 130-150 oF (54-66 oC) ~160 oF (71 oC) 5.0-5.5 Breaks down starch chains, linearly, into maltose Proteases 122-138 oF (50-59 oC) ~155 oF (68 oC) 4.6-5.3 Break down proteins o o o o (peptidase) 113-128 F (45-53 C) ~145 F (63 C) (increase FAN)

β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC) 4.5-5.5 Breaks down cell-wall materials Limit-dextrinase 95-140 oF (35-60 oC) ~150 oF (65 oC) 5.0-5.8 Breaks down sugars left behind by amylases, can be inhibited Mashing (cont’d)

Rough enzyme ranges in Mash John Palmer – How to Brew Amylases β-amylase: • Lower temp • Acts faster on larger molecules • Only works on linear starches from non-reducing end. • Produces maltose.

α-amylase: • Cuts ‘randomly’ • Works on branched amylopectin (but not at branch points.

Enzyme activity on starches Kunze Amylases (cont’d)

Brewer’s Window Jake McWhirter – Missionary Brewer Blog Limit Dextrinase Will further break down limit-dextrins.

• HOWEVER – inhibited at a very early point in the mash. Competitive inhibitor Kevin Ahern & Indira Rajagopal, Biochemistry Free & Easy, 2019 • Inhibitors act to block an enzyme from performing its job (without denaturing it).

Non-Competitive inhibitor Kevin Ahern & Indira Rajagopal, Biochemistry Free & Easy, 2019 Proteases and Glucanases

However:

Enzymes can break down the remaining cellular matrix (protein and glucan rests): Kilning denatures many of these enzymes. • Higher efficiency • Lower mash / wort viscosity Prolonged rests increases 4-vinyl- • Reduce protein haze in the beer guaiacol off flavor (ferulic acid converts • Increase FAN in whirlpool).

Also can increase viscosity. Proteases and Glucanases (cont’d) Enzyme Temperature Denatures Range α-amylase 150-160 oF (66-71 ~170 oF (77 oC) oC)

β-amylase 130-150 oF (54-66 ~160 oF (71 oC) oC)

Proteases 122-138 oF (50-59 ~155 oF (68 o o (peptidase) C) C) 113-128 oF (45-53 ~145 oF (63 oC) oC) β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC)

Limit- 95-140 oF (35-60 oC) ~150 oF (65 o dextrinase C) β-glucan Reducing Mash (poorly modified malt): Biokemist Proteases and Glucanases (cont’d) oF Enzyme Temperature Denatures 176 Range 167 α-amylase 150-160 oF (66-71 ~170 oF (77 158 o o C) C) 149 140 β-amylase 130-150 oF (54-66 ~160 oF (71 131 o o C) C) 122 113 Proteases 122-138 oF (50-59 ~155 oF (68 104 o o (peptidase) C) C) 95 113-128 oF (45-53 ~145 oF (63 86 oC) oC) β-glucanase 95-131 oF (35-55 oC) ~140 oF (60 oC)

Limit- 95-140 oF (35-60 oC) ~150 oF (65 Alternate β-glucan Reducing Mash: oC) dextrinase 1: Thin Mash // 2: Thick mash (3/4) 3: Solubilase Rest // 4: Cold Water Addition Kunze Adjuncts, Cereal Cookers, Decoction Mashing

In an infusion mash, barley malt carries the DP load. • Can support adjuncts with no amylase activity, up to roughly 20%.

• Slightly more, if the adjunct is flaked.

• Malted adjunct has some DP.

Competitive inhibitor Courtesy of Canadian Malting Barley Technical Centre Adjuncts, Cereal Cookers, Decoction mashing: Decoction Mashing (cont’d) • Solution to poor temp control. Cereal Cookers: • Gelatinize starches in adjuncts. • Portion of the mash is boiled, then added to larger mash. • Heated portion’s enzymes are • Barley malt or mash is added for α- denatured. amylase, which rapidly denatures.

• Again creating an enzymatically • A ‘lossy’ process for enzymes. ‘lossy’ process.

Brew Process Courtesy of Yuengling & Son, Inc Fermentation: Dry Hop Creep (Thanks OSU!):

Hops contain enzymes.

High rates of dry-hopping + unfiltered beer = new sugars = re-ferment.

Results: increased ABV, carb levels, and potentially more diacetyl.

Check out the CBC 2020 presentation, and look for the new white paper! Excerpt from “The Brewer’s Guardian” March 28, 1893, page 93 Questions? (ask below to be included in the recording) “Have a keen understanding of the microbiology [of brewing], because it is very Email: interesting. [email protected] No, not interesting – actually, it’s boring as shit. But it is important.”

- Professor Michael Lewis