Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO

α-amylase is an enzyme that breakdown starch or glycogen to release simple molecules such as maltose and glucose. SR74 α-amylase produced from the Geobacillus stearothermophilus SR74 has huge potential for commercialization for industrial applications due to its thermostable and thermoactive propert...

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Main Author:	Mohamad Nasir, Nurul Syazwani
Format:	Thesis
Language:	English
Published:	2018
Subjects:	Formaldehyde Enzymology Food - Composition
Online Access:	http://psasir.upm.edu.my/id/eprint/83017/1/FBSB%202018%2054%20ir.pdf
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id	my-upm-ir.83017
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spelling	my-upm-ir.830172022-01-11T03:45:36Z Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO 2018-12 Mohamad Nasir, Nurul Syazwani α-amylase is an enzyme that breakdown starch or glycogen to release simple molecules such as maltose and glucose. SR74 α-amylase produced from the Geobacillus stearothermophilus SR74 has huge potential for commercialization for industrial applications due to its thermostable and thermoactive properties. However, its expression in wild type host is too low. Thus, it was cloned and expressed in two expression systems namely, bacteria and yeast. Its expression in Escherichia coli was accompanied with the formation of inclusion bodies. The drawbacks of E. coli expression system were overcome by the Pichia pastoris expression system. However, SR74 α-amylase expression in P. pastoris under alcohol oxidase promoter (PAOX) required longer fermentation time and high methanol consumption. Therefore, there is a need for a new expression system that can overproduce α-amylase extracellularly without methanol induction with shorter fermentation time. This study aims to express SR74 α-amylase in a newly developed yeast expression system, Meyerozyma guilliermondii strain SO using formaldehyde dehydrogenase promoter (PFLD). Initially, an integration site of pFLDα in M. guilliermondii strain SO genome was predicted, followed by cloning and expression of α-amylase using PFLD. Then, the α-amylase production in M. guilliermondii strain SO was optimized. FLD gene was determined and isolated with the help of hmmer3.1b-2 (HMM) software for prediction of integration site. Based on nucleic acid sequence analysis, it was confirmed that pFLDα was able to integrate at PFLD loci in M. guilliermondii strain SO genome. Then, SR74 α-amylase gene was cloned into pFLDα followed by transformation into M. guilliermondii strain SO. Colony PCR, α-amylase screening plate and western blot analysis proved that SR74 α-amylase was successfully cloned and expressed in M. guilliermondii strain SO. Qualitative (screening plate) and quantitative screenings (Dinitrosalicylic acid assay) for recombinant α-amylase production were performed to assess the activity of recombinant α-amylase under PFLD regulation. Optimization of extracellular SR74 α-amylase production (assayed at 65 °C) in M. guilliermondii strain SO found that the highest expression was 26 U/mL at 24 h, without methanol induction. The yield was 16-fold higher than the wild-type. G. stearothermophilus SR74 and was comparable to the P. pastoris system at 5.17 times faster without methanol induction. In conclusion, the SR74 α-amylase has successfully been expressed in this newly expression with shorter cultivation time and without inducer requirements at comparable yield than established P. pastoris expression system. Shorter time and no inducer requirement were expected to significantly reduce the cost of production. The optimization procedure by Response Surface Methodology (RSM) software, mutational study and in-depth study on PFLD regulation were suggested to increase the production and to make the enzyme more favorable to industrial application. Formaldehyde Enzymology Food - Composition 2018-12 Thesis http://psasir.upm.edu.my/id/eprint/83017/ http://psasir.upm.edu.my/id/eprint/83017/1/FBSB%202018%2054%20ir.pdf text en public masters Universiti Putra Malaysia Formaldehyde Enzymology Food - Composition Oslan, Siti Nurbaya
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
advisor	Oslan, Siti Nurbaya
topic	Formaldehyde Enzymology Food - Composition
spellingShingle	Formaldehyde Enzymology Food - Composition Mohamad Nasir, Nurul Syazwani Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO
description	α-amylase is an enzyme that breakdown starch or glycogen to release simple molecules such as maltose and glucose. SR74 α-amylase produced from the Geobacillus stearothermophilus SR74 has huge potential for commercialization for industrial applications due to its thermostable and thermoactive properties. However, its expression in wild type host is too low. Thus, it was cloned and expressed in two expression systems namely, bacteria and yeast. Its expression in Escherichia coli was accompanied with the formation of inclusion bodies. The drawbacks of E. coli expression system were overcome by the Pichia pastoris expression system. However, SR74 α-amylase expression in P. pastoris under alcohol oxidase promoter (PAOX) required longer fermentation time and high methanol consumption. Therefore, there is a need for a new expression system that can overproduce α-amylase extracellularly without methanol induction with shorter fermentation time. This study aims to express SR74 α-amylase in a newly developed yeast expression system, Meyerozyma guilliermondii strain SO using formaldehyde dehydrogenase promoter (PFLD). Initially, an integration site of pFLDα in M. guilliermondii strain SO genome was predicted, followed by cloning and expression of α-amylase using PFLD. Then, the α-amylase production in M. guilliermondii strain SO was optimized. FLD gene was determined and isolated with the help of hmmer3.1b-2 (HMM) software for prediction of integration site. Based on nucleic acid sequence analysis, it was confirmed that pFLDα was able to integrate at PFLD loci in M. guilliermondii strain SO genome. Then, SR74 α-amylase gene was cloned into pFLDα followed by transformation into M. guilliermondii strain SO. Colony PCR, α-amylase screening plate and western blot analysis proved that SR74 α-amylase was successfully cloned and expressed in M. guilliermondii strain SO. Qualitative (screening plate) and quantitative screenings (Dinitrosalicylic acid assay) for recombinant α-amylase production were performed to assess the activity of recombinant α-amylase under PFLD regulation. Optimization of extracellular SR74 α-amylase production (assayed at 65 °C) in M. guilliermondii strain SO found that the highest expression was 26 U/mL at 24 h, without methanol induction. The yield was 16-fold higher than the wild-type. G. stearothermophilus SR74 and was comparable to the P. pastoris system at 5.17 times faster without methanol induction. In conclusion, the SR74 α-amylase has successfully been expressed in this newly expression with shorter cultivation time and without inducer requirements at comparable yield than established P. pastoris expression system. Shorter time and no inducer requirement were expected to significantly reduce the cost of production. The optimization procedure by Response Surface Methodology (RSM) software, mutational study and in-depth study on PFLD regulation were suggested to increase the production and to make the enzyme more favorable to industrial application.
format	Thesis
qualification_level	Master's degree
author	Mohamad Nasir, Nurul Syazwani
author_facet	Mohamad Nasir, Nurul Syazwani
author_sort	Mohamad Nasir, Nurul Syazwani
title	Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO
title_short	Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO
title_full	Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO
title_fullStr	Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO
title_full_unstemmed	Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO
title_sort	expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in meyerozyma guilliermondii strain so
granting_institution	Universiti Putra Malaysia
publishDate	2018
url	http://psasir.upm.edu.my/id/eprint/83017/1/FBSB%202018%2054%20ir.pdf
_version_	1747813337676120064

Expression of thermostable α-amylase using formaldehyde dehydrogenase promoter in Meyerozyma guilliermondii strain SO

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