Investigation of the orientation of Myriococcum thermophilum Cellobiose Dehydrogenase (MtCDH) immobilized on graphite electrodes

Abstract

Orientation of Myriococcum thermophilum Cellobiose Dehydrogenase (MtCDH) immobilized on spectroscopic graphite electrodes has been investigated electrochemically through flow injection analysis in order to obtain more information about the DET efficiency of this enzyme. The effect of applied potential and the long term stability of the modified electrodes were determined, as well as the bioelectrocatalytic current response with lactose as substrate was investigated by amperometric flow injection measurements in both direct and mediated electron transfer mode, with 1,4-benzoquinone as mediator. The effect of immobilization method on enzyme orientation was tested, and the assumption regarding orientation of MtCDH was accomplished by involving papain as restriction enzyme for cutting CDH molecules both in solution and on graphite electrodes.

Popular Abstract

In this work the enzyme called Myriococcum thermophilum Cellobiose Dehydrogenase (MtCDH) was investigated by chemical methods with a final intention to contribute on building up a device which will be used by the aid of this enzyme to measure the substrate called lactose, which is an important component in milk and many other products. But, before going in details why lactose is such an important component to be controlled, a few important words about enzymes in general, what they are, what they do, their specificity, how they work, and which things affect their activity will be mention as follows, as well as, a few facts particularly will be mention about Myriococcum thermophilum Cellobiose Dehydrogenase (MtCDH), the enzyme that I worked with in this thesis.
Enzymes are special types of proteins. Like all proteins, enzymes are made from strings of amino acids. The function of the enzyme is determined by the sequence of amino acids, types of amino acids, and the shape of the string. They are responsible for a lot of the work that is going on in cells. They act as catalysts in order to help produce and speed up chemical reactions. When a cell needs to get something done, it almost always uses an enzyme to speed things along. Enzymes are very specific. This means that each type of enzyme only reacts with the specific type of substance that it was made for. This is important so enzymes don't go around doing the wrong thing and causing chemical reactions where they are aren't supposed to. Enzymes have a special pocket on their surface called an "active site." The molecule that they are supposed to react with fits neatly right into that pocket. The molecule or substance that the enzyme reacts with is called the "substrate." The reaction takes place between the enzyme and the substrate at the active site. After the reaction is complete, the new molecule or substance is released by the enzyme. This new substance is called the "product." The environment of the enzyme and the substrate can affect the speed of the reaction. In some cases the environment can cause the enzyme to stop working or even unravel. When an enzyme stops working we call it "denatured." Here are some things that can affect enzyme activity: The temperature can affect the reaction rate. The higher the temperature, the faster the reaction will occur. However, at some point the temperature will become so high that the enzyme will denature and stop working. In many cases the pH level, or acidity, of the environment around the enzyme and substrate can affect the reaction rate. An extreme pH (high or low) will typically slow the reaction or even stop the reaction altogether. Concentration of the substrate or enzyme is another important factor. A higher concentration of substrate or enzyme can increase the reaction rate. On the other hand inhibitors are molecules that are specially made to stop the activity of enzymes. They may just slow down the reaction or stop it altogether. Some inhibitors bond with the enzyme causing it to change shape and not work correctly. The opposite of an inhibitor is an activator which can help to speed up the reaction. Enzymes don't get used up after they do their job. They can be used over and over. Enzymes are often used in industrial applications such as food processing, paper manufacturing, and detergents. There is an enzyme in human saliva called amylase that helps to break down starches as one chew. Enzymes also play an important role in breaking down our food so our bodies can use it. There are special enzymes to break down different types of foods. They are found in our saliva, stomach, pancreas, and small intestine and many other organisms as well.
The enzyme I worked with as mentioned above is called Cellobiose Dehydrogenase (CDH), produced from the ascomycete fungus Myriococcum thermophilum (Mt). The natural function of this enzyme is to substract electrons from cellobiose sugar units and deliver them to another enzyme, which makes highly reactive radicals with the help of those electrons and oxygen. Those oxygen radicals help on wood degradation.
In this thesis the CDH molecules were put on the surface of graphite electrode and instead of delivering the electrons to the radical producing protein as mention on the previous paragraph, the electrons were delivered directly to the graphite electrode and the amount of gained electrons were counted by measuring the electrical current, which is nothing else than a flow of electrons.
The device build based on such components is called a biosensor. The adsorption of CDH on the graphite electrode surface was shown to be stable and operational for a long time. Enzyme kinetics performed in this work has shown that CDH has high specificity toward lactose sugar. The influence of the applied potential on the catalytic response was investigated and results show that very little potential (mV) is needed to be applied to get the maximal oxidation current. CDH also has shown efficient direct electron transfer properties at graphite electrode toward lactose substrate. Therefore, such and many other beneficial features that CDH possess make it a promising enzyme on development of biosensors.
Regarding lactose sugar, it can be found in milk and dairy products such as cheese and yogurt, in bread and baked goods, processed breakfast cereals, instant potatoes, some soups and non-kosher lunch meats, candies, dressings and mixes for pancakes and biscuits etc. After eating such dairy products that contain this sugar, usually lactase, a digestive enzyme of the small intestine, helps to breakdown this complex sugar into two simple sugars, glucose and galactose. These simple sugars are then absorbed in the small intestine and ultimately reach the blood stream where they act as nutrients. In many cases because lactose is not digested properly in the small intestine of individuals who are lactose intolerant, it passes whole into the large intestine or colon and causes many harmful symptoms.
Eating lactose-containing products will result in discomfort for many who are lactose intolerant and this was the whole intention of this investigation to contribute on building up a device which might control this component in different products. CDH show promising features to build up such a device but much more investigations remains to be done and I look forward to give my contribution.

• author: Salihu, Ardian
• supervisor: Lo Gorton
• organization: Department of Chemistry
• course: KEMR16 20161
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: biosensor, amperometric flow injection analysis, cellobiose dehydrogenase, MtCDH, Myriococcum thermophilum, direct electron transfer, mediated electron transfer, analytical chemistry, analytisk kemi
• language: English
• id: 8875134
• date added to LUP: 2016-06-10 10:13:50
• date last changed: 2016-06-10 10:13:50