Functional Characterization of an Oat Lipase Candidate

Abstract

Oats (Avena sativa) are a valued food product due to their nutritional profile and proven health benefits. However, prior to consumption, they require heat processing to prevent off-flavors and undesirable mouthfeel caused by lipase-catalyzed hydrolysis of lipids. While the heat treatment inactivates lipases, it raises concerns regarding sustainability and oat quality. The discovery of novel functional lipases could enable engineering of heat-sensitive variants, reducing the heat required for inactivation. Additionally, lipases have broad industrial applications, and new variants could expand the use of seed lipases in the pharmaceutical, food, and biotechnology industries.

The lack of comprehensively annotated reference genomes for oats has previously limited the study of oat lipases. However, from early versions of the high-quality sequenced cv. Sang oat genome, a sequence believed to encode an oat lipase was identified. This project aimed to determine whether the identified sequence indeed encodes a functional lipase. Three versions of the sequence, one full-length and two truncated forms, were cloned into pET11a vectors and expressed in E.coli BL21 (DE3) cells under the control of the T7lac promoter. All constructs were successfully expressed as insoluble proteins. Solubilization and refolding strategies were evaluated in combination with Immobilized Metal Affinity Chromatography (IMAC). The most successful method involved solubilization in 8M urea, 300mM NaCl, 100mM NaH2PO4 (pH 7.5) buffer, followed by IMAC, and dialysis against the same buffer without urea. Lipase activity was assessed using a 4-Methylumbelliferyl heptanoate (4-MUH) assay. Activity was detected for the shortest truncated version, suggesting that the identified sequence may indeed encode a functional lipase. However, due to measurement uncertainties and the absence of detectable activity for the two other versions, further experiments are required to validate the lipase activity of the shortest version and assess potential activity in the remaining two.

Popular Abstract

Unexplored Oat Proteins: Towards Better Processing and New Industrial Uses
Oats are not just a popular food, they also contain important proteins called lipases. By studying these oat lipases, we could make oat processing more sustainable and open the door to new industrial lipase applications.

Do you follow a vegetarian, vegan, gluten-free, or health-conscious diet? If so, you are probably familiar with oats. Thanks to their high protein content, cholesterol-lowering effects, anti-inflammatory properties and naturally gluten-free nature, they have become a popular food product. However, before oats can be consumed as a food product, they must be processed.

One key step in oat processing is the inactivation of lipases, enzymes (proteins) that break down fats into smaller components called fatty acids. While lipase activity is essential for the growth of the oat seed, it can lead to unpleasant flavors and shorten the shelf life of oat products. The current solution is heat treatment. This inactivates the lipases, however, it is an energy intensive process that can negatively affect the oat quality.

Interestingly, lipases are not just a challenge in oat processing, they are also important in many industries, ranging from detergents to pharmaceuticals. A better understanding of oat lipases could broaden their industrial applications or enable engineering of more heat-sensitive variants, potentially making oat processing more sustainable. The current challenge is that oat lipases remain poorly understood.
In this project, I investigated a gene, a set of instructions for making a protein, that is believed to encode an oat lipase. My goal was to determine whether this gene actually encodes a functional lipase. To test this, I used a technique called recombinant protein production, which is kind of like copy-and-paste. I copied three versions of the gene in the lab and inserted them into bacteria. These bacteria act as protein factories, using the inserted instructions to produce the proteins. After production, the proteins were purified, meaning they were separated from the bacterial components to obtain clean samples.

To find out if the proteins were functional lipases, I used a fat-like substrate. When broken down by a lipase and exposed to UV light, this substrate releases a molecule that gives off blue light. I observed blue light from one of the three gene variants, suggesting it does encode a functional lipase. However, the other two variants did not show any blue light, so further experiments are needed to confirm whether the protein is truly an oat lipase.

So, even if you are already familiar with oats in your everyday meals, they also contain important proteins. With more research, we might be able to engineer these lipases for better oat processing or new industrial uses.

• author: Lundquist, Emma
• supervisor: Sofia Marmon; Johan Svensson Bonde
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20251
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences; Technology and Engineering
• keywords: oats, lipase, lipase activity, recombinant protein expression, applied biochemistry
• language: English
• id: 9192706
• date added to LUP: 2025-06-13 11:35:14
• date last changed: 2025-06-13 11:35:14