Cargando…

Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell

BACKGROUND: β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted...

Descripción completa

Detalles Bibliográficos
Autores principales: Chang, Fei, Zhang, Xianbing, Pan, Yu, Lu, Youxue, Fang, Wei, Fang, Zemin, Xiao, Yazhong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688802/
https://www.ncbi.nlm.nih.gov/pubmed/29115967
http://dx.doi.org/10.1186/s12896-017-0402-1
_version_ 1783279244004032512
author Chang, Fei
Zhang, Xianbing
Pan, Yu
Lu, Youxue
Fang, Wei
Fang, Zemin
Xiao, Yazhong
author_facet Chang, Fei
Zhang, Xianbing
Pan, Yu
Lu, Youxue
Fang, Wei
Fang, Zemin
Xiao, Yazhong
author_sort Chang, Fei
collection PubMed
description BACKGROUND: β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance. RESULTS: Light activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C. CONCLUSIONS: We have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-017-0402-1) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5688802
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-56888022017-11-24 Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell Chang, Fei Zhang, Xianbing Pan, Yu Lu, Youxue Fang, Wei Fang, Zemin Xiao, Yazhong BMC Biotechnol Research Article BACKGROUND: β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance. RESULTS: Light activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C. CONCLUSIONS: We have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-017-0402-1) contains supplementary material, which is available to authorized users. BioMed Central 2017-11-07 /pmc/articles/PMC5688802/ /pubmed/29115967 http://dx.doi.org/10.1186/s12896-017-0402-1 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Chang, Fei
Zhang, Xianbing
Pan, Yu
Lu, Youxue
Fang, Wei
Fang, Zemin
Xiao, Yazhong
Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell
title Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell
title_full Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell
title_fullStr Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell
title_full_unstemmed Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell
title_short Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell
title_sort light induced expression of β-glucosidase in escherichia coli with autolysis of cell
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688802/
https://www.ncbi.nlm.nih.gov/pubmed/29115967
http://dx.doi.org/10.1186/s12896-017-0402-1
work_keys_str_mv AT changfei lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell
AT zhangxianbing lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell
AT panyu lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell
AT luyouxue lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell
AT fangwei lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell
AT fangzemin lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell
AT xiaoyazhong lightinducedexpressionofbglucosidaseinescherichiacoliwithautolysisofcell