Study on putative lipases from Bifidobactirium longum and Lactobacillus rhamnosus
(2020) KLGM01 20181Food Technology and Nutrition (M.Sc.)
- Abstract
- Lipases are the main fat digestive enzymes present in the digestive system of many animals, microorganisms and plants. Probiotic microorganisms that can possibly produce enzymes are of crucial importance since it can have an effect on human health. Two of the main probiotic strains are Lactobacillus rhamnosus and Biffidobacterium longum, in each there was a putative lipase encoded on the DNA and there is lack of information on these two putative lipases. Both strains are Gram-positive and anaerobic which adds an extra difficulty on the production of the enzymes. For that reason, recombinant production of the enzymes was chosen.
Recombinant production was carried out at first with three strains of E. coli to establish the best one for the... (More) - Lipases are the main fat digestive enzymes present in the digestive system of many animals, microorganisms and plants. Probiotic microorganisms that can possibly produce enzymes are of crucial importance since it can have an effect on human health. Two of the main probiotic strains are Lactobacillus rhamnosus and Biffidobacterium longum, in each there was a putative lipase encoded on the DNA and there is lack of information on these two putative lipases. Both strains are Gram-positive and anaerobic which adds an extra difficulty on the production of the enzymes. For that reason, recombinant production of the enzymes was chosen.
Recombinant production was carried out at first with three strains of E. coli to establish the best one for the production. Production of the lipases was possible with the E. coli BL21 (DE3) strain and the same strain was used to test the possible bioreactor scale production using a 2.5 L counter top bioreactor. Once the best strain was chosen; the production in lab scale was tried and then purification of the lipases was tested with Ion Metal Affinity Chromatography (IMAC). With established production and purification, the kinetic characterization of the putative lipases was carried out and it included calculation of the activity constants, determination of optimal temperature and pH and activity over long storage time. Oligomerization of the lipases on the optimal conditions was tested using Asymmetric flow-field flow fractionation (AF4).
The production of the lipases was successful in both lab scale and bioreactor scale and then the characterization was conducted on those lipases produced by the trials. Activity was measured and the kinetic parameters were established showing substrate inhibition for the Lactobacillus rhamnosus (LR) lipase while the Biffidobacterium longum (BL) lipase never reached the substrate saturation plateau. The optimal conditions were determined and they were matching the physiological conditions of pH 6-7 and temperature of 30°C - 40°C. Storage time at 37°C was established and the LR lipase had a half time of 3 days while the BL lipase was not stable at that temperature. From AF4 analysis both lipases seem to be as monomers in the optimal conditions but the results are not conclusive. (Less) - Popular Abstract
- Lipases are enzymes which catalyse the degradation of triglycerides. Due to this function, they play a crucial role in fat digestion, and therefore in the metabolic implications of excessive consumption of fat. Indeed, this is the principal reason for many chronic diseases like obesity, diabetes and various types of cancer. Lipases can be found in any animal, plant or microorganism. Microbial lipases are of interest because of the increasing use of microorganism as functional food ingredients (probiotics).Probiotics are bacteria that exert health benefits for humans and animals (FAO,2001). Two of the most studied and used probiotic bacteria are Lactobacillus rhamnosus (LR) and Biffidobacterium longum (BL). However, from literature research... (More)
- Lipases are enzymes which catalyse the degradation of triglycerides. Due to this function, they play a crucial role in fat digestion, and therefore in the metabolic implications of excessive consumption of fat. Indeed, this is the principal reason for many chronic diseases like obesity, diabetes and various types of cancer. Lipases can be found in any animal, plant or microorganism. Microbial lipases are of interest because of the increasing use of microorganism as functional food ingredients (probiotics).Probiotics are bacteria that exert health benefits for humans and animals (FAO,2001). Two of the most studied and used probiotic bacteria are Lactobacillus rhamnosus (LR) and Biffidobacterium longum (BL). However, from literature research there are no available information on lipases from probiotics. Interestingly, genomes of L rhamnosus and B. longum, which inhabit in the human gut, encode potential lipases, wich biological function is unknown.
The aim of this thesis is to investigate the activity of the potential lipases mentioned above and characterize their physical chemistry properties. To start with, the recombinant production of the lipases was used. Recombinant production is when the enzymes are produced in another microorganism other than the original. This was performed in Escherichia coli as host because the conditions at which these lipases are produced by the original microorganism are not established. The characterization included the determination of optimal pH-temperature, kinetic parameters, stability of storage time, and structural analysis of potential oligomeric forms and aggregates. All the reactions were performed using the synthetic substrate paranitrophenol-laurate.
The results obtained for the optimal conditions showed that both lipases have higher reaction rates at pH 6 and temperature ranging from 30°C to 40°C. By knowing the optimal conditions, the reaction rate of the lipases was established by exposing them to increasing amounts of substrate and recording the amount of resulting product. The LR lipase showed substrate inhibition, which means that as the substrate concentration was increasing the lipase, was becoming less and less active. On the other hand, the BL lipase had increasing reaction rate over the whole range of substrate concentrations used. Storage time of both lipases was examined and the LR showed a decreasing reaction rate while the BL lipase was not stable when exposed for long periods in the storage temperature. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9005495
- author
- Manasian, Panagiotis LU
- supervisor
- organization
- course
- KLGM01 20181
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- food technology, livsmedelsteknologi
- language
- English
- id
- 9005495
- date added to LUP
- 2020-04-17 14:36:31
- date last changed
- 2020-04-17 14:36:31
@misc{9005495,
abstract = {{Lipases are the main fat digestive enzymes present in the digestive system of many animals, microorganisms and plants. Probiotic microorganisms that can possibly produce enzymes are of crucial importance since it can have an effect on human health. Two of the main probiotic strains are Lactobacillus rhamnosus and Biffidobacterium longum, in each there was a putative lipase encoded on the DNA and there is lack of information on these two putative lipases. Both strains are Gram-positive and anaerobic which adds an extra difficulty on the production of the enzymes. For that reason, recombinant production of the enzymes was chosen.
Recombinant production was carried out at first with three strains of E. coli to establish the best one for the production. Production of the lipases was possible with the E. coli BL21 (DE3) strain and the same strain was used to test the possible bioreactor scale production using a 2.5 L counter top bioreactor. Once the best strain was chosen; the production in lab scale was tried and then purification of the lipases was tested with Ion Metal Affinity Chromatography (IMAC). With established production and purification, the kinetic characterization of the putative lipases was carried out and it included calculation of the activity constants, determination of optimal temperature and pH and activity over long storage time. Oligomerization of the lipases on the optimal conditions was tested using Asymmetric flow-field flow fractionation (AF4).
The production of the lipases was successful in both lab scale and bioreactor scale and then the characterization was conducted on those lipases produced by the trials. Activity was measured and the kinetic parameters were established showing substrate inhibition for the Lactobacillus rhamnosus (LR) lipase while the Biffidobacterium longum (BL) lipase never reached the substrate saturation plateau. The optimal conditions were determined and they were matching the physiological conditions of pH 6-7 and temperature of 30°C - 40°C. Storage time at 37°C was established and the LR lipase had a half time of 3 days while the BL lipase was not stable at that temperature. From AF4 analysis both lipases seem to be as monomers in the optimal conditions but the results are not conclusive.}},
author = {{Manasian, Panagiotis}},
language = {{eng}},
note = {{Student Paper}},
title = {{Study on putative lipases from Bifidobactirium longum and Lactobacillus rhamnosus}},
year = {{2020}},
}