Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris
Abstract Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries....
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doaj-440e9b4bff16445cac516eece83b93cf2020-11-25T03:13:22ZengBMCBMC Research Notes1756-05002019-09-011211610.1186/s13104-019-4638-9Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastorisC. G. Conacher0M. P. García-Aparicio1G. Coetzee2W. H. van Zyl3J. F. Gӧrgens4Departments of Process Engineering, Stellenbosch UniversityDepartments of Process Engineering, Stellenbosch UniversityDepartments of Process Engineering, Stellenbosch UniversityDepartments of Microbiology, Stellenbosch UniversityDepartments of Process Engineering, Stellenbosch UniversityAbstract Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application.http://link.springer.com/article/10.1186/s13104-019-4638-9Glucuronoyl esteraseCellobiose dehydrogenaseMethanol-free constitutive expression systemPichia pastorisLignocellulose biorefinery enzymesEnzyme bioprocess technology |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
C. G. Conacher M. P. García-Aparicio G. Coetzee W. H. van Zyl J. F. Gӧrgens |
spellingShingle |
C. G. Conacher M. P. García-Aparicio G. Coetzee W. H. van Zyl J. F. Gӧrgens Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris BMC Research Notes Glucuronoyl esterase Cellobiose dehydrogenase Methanol-free constitutive expression system Pichia pastoris Lignocellulose biorefinery enzymes Enzyme bioprocess technology |
author_facet |
C. G. Conacher M. P. García-Aparicio G. Coetzee W. H. van Zyl J. F. Gӧrgens |
author_sort |
C. G. Conacher |
title |
Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris |
title_short |
Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris |
title_full |
Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris |
title_fullStr |
Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris |
title_full_unstemmed |
Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris |
title_sort |
scalable methanol-free production of recombinant glucuronoyl esterase in pichia pastoris |
publisher |
BMC |
series |
BMC Research Notes |
issn |
1756-0500 |
publishDate |
2019-09-01 |
description |
Abstract Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application. |
topic |
Glucuronoyl esterase Cellobiose dehydrogenase Methanol-free constitutive expression system Pichia pastoris Lignocellulose biorefinery enzymes Enzyme bioprocess technology |
url |
http://link.springer.com/article/10.1186/s13104-019-4638-9 |
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