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Functional expression of a novel methanol-stable esterase by Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system
Wei Wei
East China University of Science and Technology
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at BMC Biotechnology.
Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing.
Results: The gene estGSU 753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of p NP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate p NP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol . After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M).
Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.
Keywords
Geobacillus subterraneus, esterase, methanol-stable, biocatalysis, cinnamyl acetate
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CURRENT STATUS: Published
Version 5
Posted 12 May, 2020
Published
On 29 Jun, 2020
No community comments so far
Editorial decision: Accept
On 12 May, 2020
Submission checks complete
On 27 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 06 Apr, 2020
Editor assigned
On 05 Apr, 2020
Submission checks complete
On 04 Apr, 2020
Editor invited
On 04 Apr, 2020
No comments provided
Review #1 received
Received 24 Mar, 2020
Editorial decision: Minor revision
On 24 Mar, 2020
Reviewer #2 agreed
On 13 Mar, 2020
Review #2 received
Received 13 Mar, 2020
Reviewer #1 agreed
On 11 Mar, 2020
Reviewers invited
Invitations sent on 10 Mar, 2020
Editor assigned
On 08 Mar, 2020
Editor invited
On 07 Mar, 2020
Submission checks complete
On 05 Mar, 2020
No comments provided
Review #1 received
Received 23 Feb, 2020
Editorial decision: Minor revision
On 23 Feb, 2020
Review #2 received
Received 17 Feb, 2020
Review #3 received
Received 17 Feb, 2020
Reviewer #3 agreed
On 15 Feb, 2020
Reviewer #2 agreed
On 09 Feb, 2020
Reviewer #1 agreed
On 04 Feb, 2020
Reviewers invited
Invitations sent on 19 Dec, 2019
Editor assigned
On 03 Dec, 2019
Submission checks complete
On 02 Dec, 2019
Editor invited
On 02 Dec, 2019
No comments provided
Editorial decision: Minor revision
On 20 Nov, 2019
Review #2 received
Received 09 Nov, 2019
Review #1 received
Received 27 Oct, 2019
Reviewer #2 agreed
On 17 Oct, 2019
Reviewers invited
Invitations sent on 14 Oct, 2019
Reviewer #1 agreed
On 14 Oct, 2019
Editor assigned
On 29 Sep, 2019
Submission checks complete
On 28 Sep, 2019
Editor invited
On 28 Sep, 2019
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Subject Areas
Biotechnology and Bioengineering
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This preprint is under consideration at BMC Biotechnology. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Functional expression of a novel methanol-stable esterase by Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system
Wei Wei
East China University of Science and Technology
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 5
Posted 12 May, 2020
Published
On 29 Jun, 2020
No community comments so far
Editorial decision: Accept
On 12 May, 2020
Submission checks complete
On 27 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 06 Apr, 2020
Editor assigned
On 05 Apr, 2020
Submission checks complete
On 04 Apr, 2020
Editor invited
On 04 Apr, 2020
No comments provided
Review #1 received
Received 24 Mar, 2020
Editorial decision: Minor revision
On 24 Mar, 2020
Reviewer #2 agreed
On 13 Mar, 2020
Review #2 received
Received 13 Mar, 2020
Reviewer #1 agreed
On 11 Mar, 2020
Reviewers invited
Invitations sent on 10 Mar, 2020
Editor assigned
On 08 Mar, 2020
Editor invited
On 07 Mar, 2020
Submission checks complete
On 05 Mar, 2020
No comments provided
Review #1 received
Received 23 Feb, 2020
Editorial decision: Minor revision
On 23 Feb, 2020
Review #2 received
Received 17 Feb, 2020
Review #3 received
Received 17 Feb, 2020
Reviewer #3 agreed
On 15 Feb, 2020
Reviewer #2 agreed
On 09 Feb, 2020
Reviewer #1 agreed
On 04 Feb, 2020
Reviewers invited
Invitations sent on 19 Dec, 2019
Editor assigned
On 03 Dec, 2019
Submission checks complete
On 02 Dec, 2019
Editor invited
On 02 Dec, 2019
No comments provided
Editorial decision: Minor revision
On 20 Nov, 2019
Review #2 received
Received 09 Nov, 2019
Review #1 received
Received 27 Oct, 2019
Reviewer #2 agreed
On 17 Oct, 2019
Reviewers invited
Invitations sent on 14 Oct, 2019
Reviewer #1 agreed
On 14 Oct, 2019
Editor assigned
On 29 Sep, 2019
Submission checks complete
On 28 Sep, 2019
Editor invited
On 28 Sep, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at BMC Biotechnology.
Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing.
Results: The gene estGSU 753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of p NP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate p NP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol . After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M).
Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5