Optimizing protein expression of BoGal36A

Abstract

Achieving pure protein with high enzymatic activity for research and practical teaching purposes is essential. In this study the protein BoGal36A was purified using cells or lysate obtained from three different expression methods. The expression was performed using bioreactor, shaking flasks and a prepared lysate of cells grown in shaking flasks at Lund protein Production Platform (LP3; (kindly provided by Carmen Ebenwaldner, PhD-student, Lund University). The aim was to compare which method yielded highest amount of enzyme with highest catalytic activity. A BoGal36A mutant was also expressed based on the result obtained in the study.
BoGal36A catalyzes the hydrolysis of α-galactosidic bonds and has an optimal activity at pH=6.00 and 37 ℃. The enzymes were purified using immobilized metal ion affinity chromatography (IMAC) and Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis was used to analyse the purity. The enzymatic activity was measured using 4-Nitrophenyl α-D-galactopyranoside as the substrate to determine their kinetic parameters.
The results showed that the expression of BoGal36A in bioreactors yields protein having highest enzymatic activity and catalytic efficiency compared to the other expression methods, due to the method having a controlled process and allowing higher cell densities in the induction phase. The mutant variant expressed under the same condition showed lower activity which was expected.
The conclusion of the study is that the bioreactor method is preferable before traditional cultivation in shaking flask to achieve high yield of enzyme with high catalytic activity.

Popular Abstract

Large amounts of enzymes for research and education

Enzymes are involved in chemical reactions in all living organisms. They are vital for the human body, in the medicine and how the food is proceeded. In this project the enzyme BoGal36A which breaks down sugars is studied.
The project was performed with the intention of finding out the best way to grow bacteria to produce a lot of this enzyme in a functional form. Two cultivation methods were compared, the first was growing bacteria in shaking flasks which are simply bottles placed on a platform that shakes the flasks to supply the necessary aeration. The second method was using a bioreactor which is more advanced and control temperature, pH as well as dissolved oxygen. A mutant type of BoGal36A was also tested to see how the mutation affects the enzyme function.
The process was to grow bacteria, collecting cells, break them, extract the soluble proteins and purifying the enzyme. To check if the protein was pure a gel was used to separate the different proteins in the sample, before measuring how active the enzyme was in the different preparations.
It was clear that the bioreactor method was much better than the other method. It produced more cells and enzyme, and enzyme had higher activity than the enzyme made in the shaking flasks. The mutant version had lower activity than the original BoGal36A, which matched the expectations.
The results of this project show that bioreactors are much better for producing enzymes with high activity. This knowledge can help scientists obtain large quantities of enzymes for structural and functional studies which may allow design of more effective enzymes that for example could find a use in industry such as biotechnology.

• author: Ismail, Sara
• supervisor: Urban Johanson
• organization: Department of Chemistry
• course: KEMK10 20251
• year: 2025
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: Bioreactor, BoGal36A, Shaking flasks, Expression, biochemistry
• language: English
• id: 9192948
• date added to LUP: 2025-06-05 13:23:17
• date last changed: 2025-06-05 13:23:17