Bio 1406 Determining Enzymatic Specificity and Cofactors in Latase

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Determining Enzymatic Specificity and Cofactors in Lactase

Collin College

Introduction

Lactase is an enzyme that breaks down lactose. Enzymes are proteins. Lactose is the sugar found in milk. The body readily produces lactase as a baby to help digest breastmilk, which contains lactose. From the ages of two to five the production of lactase begins to decrease in lactose intolerant people (Dzialanski, et al., 2016). By age nine, the body has completely ceased producing the enzyme. The food industry also produces lactase commercially to aid in the reduction of lactose in milk products (Talbert & Goddard, 2013). Another term for lactose intolerance is lactase non-persistence or LNP. In laymen’s terms, it is the condition in which the body does not produce any or an insufficient amount of the enzyme lactase. Maltose is the sugar found in malt. Also known as malt sugar. It is molecularly very similar to lactose. This lab experiment tested the ability of the enzyme lactase’s ability to interact and bond with the substrates maltose and lactose. Substrates are substances that enzymes act on. If knowing that the enzyme lactase is specific for the substrate lactose, then it should bond and interact better with lactose than maltose.

Lactase has been shown to retain high amounts of cofactors. Cofactors are ions or non-protein molecules that bind to enzymes (Reece, et al., 2014). When cells are disrupted, metal ions such as calcium and magnesium are frequently present. This is especially true in the enzyme lactase. In order to remove these metal ions and slow down enzymatic reactions, a chelating agent is often added. Chelating agents are chemicals that react with and cleave to metals in an enzyme. Essentially removing the cofactor from the enzyme. EDTA, ethylenediaminetetraacetic acid, is the most common chelating agent (Cain, Cardenass, & Donald-Whitney, 2008). This lab experiment tested the role that metal ions have on the enzymatic activity of lactase and if it requires a cofactor. If the chelating agent EDTA is introduced into lactase, then the effectiveness of lactase is greatly diminished.

Materials and Methods

A clean plastic pipette was used to fill one microfuge tube with 0.5 mL of milk (lactose) and 0.5 mL of maltose solution was added to a different microfuge tube using an alternate clean plastic pipette. Lactase solution was then added to each tube using a clean pipette bringing the mixture level to 1.0 mL. Both tubes were then placed in a 40° C water bath for 10 minutes. After ten minutes a glucose strip was placed in each tube for one second and then set on the bench for 30 seconds. The amount