Enhancing recombinant T1 lipase production in Pichia guilliermondii
Lipases are hydrolytic enzymes, ranked as third most relevant industrial enzymes with 5 % share in global enzyme market. Thermostable T1 lipase from Geobacillus zalihae was previously expressed under the regulatory control of alcohol oxidase promoter 1 (AOXp 1) in the methylotrophic yeast Pichia...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2017
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/70201/1/FBSB%202017%2019%20-%20IR.pdf |
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| Summary: | Lipases are hydrolytic enzymes, ranked as third most relevant industrial enzymes with
5 % share in global enzyme market. Thermostable T1 lipase from Geobacillus zalihae
was previously expressed under the regulatory control of alcohol oxidase promoter 1
(AOXp 1) in the methylotrophic yeast Pichia guilliermondii, isolated from spoiled
orange. Methanol was found to be a vital compound to induce the promoter activity in
Pichia pastoris. In this study P. guilliermondii has shown the potential to express the
recombinant lipase without methanol under the regulation of AOXp 1. This study
sought to optimise medium conditions of thermostable lipase with and without
methanol as inducer. The expression of T1 lipase without methanol was expected to
reduce cost and toxicity effect of methanol.
Buffered and non-buffered media compositions were studied for T1 lipase production,
the media were first, supplemented with methanol then without methanol. Buffer
complex methanol medium was observed to be optimum for T1 lipase production with
a 3-fold increase over non-buffered methanol medium. One-factor-at-a-time,
conventional method of optimisation was used to identify significant data range for
medium parameters. Using the observed data range, eight parameters which includes
temperature, pH, inoculum size, biomass concentration, incubation time, shaking
speed, culture volume and methanol concentration, were screened for lipase
production in methanol medium using Plackett-Burman Design. Temperature,
inoculum size, culture volume and incubation time, were observed to exert significant
effect on lipase production. These parameters were optimised using Box-Behnken
Design of Response Surface Methodology. Optimum levels of these parameters were
predicted at temperature 34 oC, culture volume 190 mL, inoculum size 4 v/v and
incubation time 24 h with an experimented lipase activity of 9.26 U/mL. Over 2-fold
increase before optimization in methanol medium was observed and 6-fold increase
over previous research work. On the other hand, six parameters which include temperature, pH, inoculum size,
incubation time, shaking speed and culture volume were screened for T1 lipase
production in medium without methanol. Three parameters were observed to have
significant effect on lipase production, then, these parameters were further optimised
using Box-Behnken Design and their optimum levels were achieved at pH of 6,
inoculum size 2 v/v and incubation time of 24 h as was predicted and the experimented
lipase activity of 2.012 U/mL was observed. This result gave 4-fold increase over
lipase production before optimisation in medium without methanol. Recombinant T1
lipase production was further scaled up to 3 L in the bioreactor for 128 h in methanol
medium and lipase activity observed was 12 U/mL at 120 h incubation time.
Meanwhile, scale up in medium without methanol yielded a lipase activity of 3.5
U/mL at 118 h incubation time. The result has proven that, the time taken for optimum
lipase production without methanol was faster from the production with methanol in
the bioreactor.
In conclusion, temperature, pH, inoculum size, incubation time and culture volume for
recombinant T1 lipase production were optimised in shake-flask and applied to the
bioreactor level. Lipase was also produced in medium without methanol both at the
shake flask level and the bioreactor. Thus, medium parameters optimisation has
proven to be very useful in enhancing thermostable lipase production in recombinant
P. guilliermondii. Although T1 lipase has been expressed in medium without methanol
but it is lower compared to medium with methanol. |
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