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A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens
Extracellular α-amylase from Pyrococcus furiosus (PFA) shows great starch-processing potential for industrial application due to its thermostability, long half-life and optimal activity at low pH; however, it is difficult to produce in large quantities. In contrast, α-amylase from Bacillus amyloliqu...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768087/ https://www.ncbi.nlm.nih.gov/pubmed/26916714 http://dx.doi.org/10.1038/srep22229 |
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author | Wang, Ping Wang, Peili Tian, Jian Yu, Xiaoxia Chang, Meihui Chu, Xiaoyu Wu, Ningfeng |
author_facet | Wang, Ping Wang, Peili Tian, Jian Yu, Xiaoxia Chang, Meihui Chu, Xiaoyu Wu, Ningfeng |
author_sort | Wang, Ping |
collection | PubMed |
description | Extracellular α-amylase from Pyrococcus furiosus (PFA) shows great starch-processing potential for industrial application due to its thermostability, long half-life and optimal activity at low pH; however, it is difficult to produce in large quantities. In contrast, α-amylase from Bacillus amyloliquefaciens (BAA) can be produced in larger quantities, but shows lower stability at high temperatures and low pH. Here, we describe a BAA protein expression pattern-mimicking strategy to express PFA in B. amyloliquefaciens using the expression and secretion elements of BAA, including the codon usage bias and mRNA structure of gene, promoter, signal peptide, host and cultivation conditions. This design was assessed to be successful by comparing the various genes (mpfa and opfa), promoters (PamyA and P43), and strains (F30, F31, F32 and F30-∆amyA). The final production of PFA yielded 2714 U/mL, about 3000- and 14-fold that reportedly produced in B. subtilis or E. coli, respectively. The recombinant PFA was optimally active at ~100 °C and pH 5 and did not require Ca(2+) for activity or thermostability, and >80% of the enzyme activity was retained after treatment at 100 °C for 4 h. |
format | Online Article Text |
id | pubmed-4768087 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-47680872016-03-02 A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens Wang, Ping Wang, Peili Tian, Jian Yu, Xiaoxia Chang, Meihui Chu, Xiaoyu Wu, Ningfeng Sci Rep Article Extracellular α-amylase from Pyrococcus furiosus (PFA) shows great starch-processing potential for industrial application due to its thermostability, long half-life and optimal activity at low pH; however, it is difficult to produce in large quantities. In contrast, α-amylase from Bacillus amyloliquefaciens (BAA) can be produced in larger quantities, but shows lower stability at high temperatures and low pH. Here, we describe a BAA protein expression pattern-mimicking strategy to express PFA in B. amyloliquefaciens using the expression and secretion elements of BAA, including the codon usage bias and mRNA structure of gene, promoter, signal peptide, host and cultivation conditions. This design was assessed to be successful by comparing the various genes (mpfa and opfa), promoters (PamyA and P43), and strains (F30, F31, F32 and F30-∆amyA). The final production of PFA yielded 2714 U/mL, about 3000- and 14-fold that reportedly produced in B. subtilis or E. coli, respectively. The recombinant PFA was optimally active at ~100 °C and pH 5 and did not require Ca(2+) for activity or thermostability, and >80% of the enzyme activity was retained after treatment at 100 °C for 4 h. Nature Publishing Group 2016-02-26 /pmc/articles/PMC4768087/ /pubmed/26916714 http://dx.doi.org/10.1038/srep22229 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Wang, Ping Wang, Peili Tian, Jian Yu, Xiaoxia Chang, Meihui Chu, Xiaoyu Wu, Ningfeng A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens |
title | A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens |
title_full | A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens |
title_fullStr | A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens |
title_full_unstemmed | A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens |
title_short | A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens |
title_sort | new strategy to express the extracellular α-amylase from pyrococcus furiosus in bacillus amyloliquefaciens |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768087/ https://www.ncbi.nlm.nih.gov/pubmed/26916714 http://dx.doi.org/10.1038/srep22229 |
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