Team:UofGuelph/Design

Project Design

Background on Beerstone, FRC and OXC

Beerstone is a salt precipitate composed primarily of calcium oxalate (C₂CaO₄). It poses a large problem in the brewing industry due to its high insolubility and use of corrosive chemicals for its effective removal from brewing equipment¹.

The reason for the high insolubility of beerstone is because one of its major components, calcium oxalate (C₂CaO₄), contains a chelator. Calcium ions in the water react with oxalic acids present in malt, forming calcium oxalate. When polypeptides found in beer are incorporated into the oxalate structure, the precipitate that is formed is known as beerstone^2,3. Geographic regions that contain high levels of calcium in their drinking water, such as Guelph, Ontario, Canada, can lead to 165g of C₂CaO₄ buildup per 1000L batch of beer⁴. The porous nature of beerstone scale promotes biofilm formation from the microorganisms present in the brewing solution. Biofilm growth causes both “off flavours” that can ruin an entire batch of beer and also be a potential biosafety hazard for the consumer⁵.

Oxalobacter formigenes is a human gut bacterium that derives its energy solely from the metabolization of oxalate using enzymes Formyl-Coenzyme A Transferase (FRC) and Oxalyl-Coenzyme A Decarboxylase (OXC). Oxalate is brought into the cell by an oxalate-formate antiporter (OxIT) and converted to CO2 and formyl-CoA. The formyl- CoA is reused by FRC as a CoA donor in a subsequent reaction and released from the cell as formate by OxIT⁶.

Objectives

- Express FRC and OXC in E. coli BL21 using pET28a vector. - Assess the feasibility of using these enzymes as an alternate cleaning method to degrade beerstone.

Project Overview

Step 1: Cloning of frc and oxc into DH5α
-Synthesize frc and oxc
-Add PstI cut site to pET-28a
-Ligate frc and oxc into pET-28a
-Transform pET-28afrc/oxc into DH5α

Step 2: Clone frc and oxc into BL21
-Purify pET-28afrc/oxc from DH5α
-Transform pET-28afrc/oxc into BL21

Step 3: Express and Purify FRC and OXC
-Induce expression with IPTG and extract crude proteins
-Purify proteins using Ni-NTA chromatography

Step 4: Characterize FRC and OXC
-Characterize enzyme function using Sodium Oxalate
-Characterize enzyme function using Calcium Oxalate

Step 5: Design a Cleaning Solution and Test on Beerstone
-Test ability of enzymes to break down Beerstone
-Design a functional cleaning solution

University of Guelph iGEM 2018