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Honghua Jia
Nanjing Tech University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3824-7768
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In present study, we have mined a ω-transaminase (ω-TA) from Chloroflexi bacterium from genome database by using an ω-TA sequence ATA117 Arrmut11 from Arthrobacter sp . KNK168 and an amine transaminase from Aspergillus terreus as templates in a BLASTP search and motif sequence alignment. The protein sequence of the ω-TA from C. bacterium shows 38% sequence identity to ATA117 Arrmut11. The gene sequence of the ω-TA was inserted into pRSF-Duet1 and functionally expressed in E. coli BL21(DE3). Results showed that the recombinant ω-TA has a specific activity of 1.19 U/mg at pH 8.5, 40 °C. The substrate acceptability test showed that ω-TA has significant reactivity to aromatic amino donors and amino receptors. More importantly, the ω-TA also exhibited a good affinity towards some cyclic substrates such as 1-Boc-3-piperidone. The homology model of the ω-TA was built by Discovery Studio and docking was performed to describe the relative activity towards some substrates. The ω-TA was evolved by site-saturation mutagenesis and found that the Q192G mutant increased the activity to the (R)-α-methylbenzylamine (MBA) by around seven-fold. The Q192G mutant was then used to convert two cyclic ketones, N -Boc-3-pyrrolidinone and N -Boc-3-aminopiperidine, and the conversions were both improved compared to the parental ω-TA.
Keywords
ω-Transaminase,Chloroflexibacterium, Characterization,Substrate specificity, Site-saturationmutagenesis
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Received 01 Apr, 2021
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On 27 Mar, 2021
Reviewer #3 agreed
On 23 Mar, 2021
Reviewer #2 agreed
On 22 Mar, 2021
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Received 22 Mar, 2021
Reviewer #1 agreed
On 22 Mar, 2021
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Invitations sent on 22 Mar, 2021
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On 11 Mar, 2021
Editor invited
On 11 Mar, 2021
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On 09 Mar, 2021
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This preprint is under consideration at Bioresources and Bioprocessing. 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
Honghua Jia
Nanjing Tech University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3824-7768
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: Under Review
Version 1
Posted 17 Mar, 2021
No community comments so far
Review #2 received
Received 13 Apr, 2021
Review #4 received
Received 11 Apr, 2021
Review #1 received
Received 10 Apr, 2021
Review #3 received
Received 01 Apr, 2021
Reviewer #4 agreed
On 27 Mar, 2021
Reviewer #3 agreed
On 23 Mar, 2021
Reviewer #2 agreed
On 22 Mar, 2021
Review #? received
Received 22 Mar, 2021
Reviewer #1 agreed
On 22 Mar, 2021
Reviewers invited
Invitations sent on 22 Mar, 2021
Editor assigned
On 11 Mar, 2021
Editor invited
On 11 Mar, 2021
Submission checks complete
On 09 Mar, 2021
First submitted
On 04 Mar, 2021
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
In present study, we have mined a ω-transaminase (ω-TA) from Chloroflexi bacterium from genome database by using an ω-TA sequence ATA117 Arrmut11 from Arthrobacter sp . KNK168 and an amine transaminase from Aspergillus terreus as templates in a BLASTP search and motif sequence alignment. The protein sequence of the ω-TA from C. bacterium shows 38% sequence identity to ATA117 Arrmut11. The gene sequence of the ω-TA was inserted into pRSF-Duet1 and functionally expressed in E. coli BL21(DE3). Results showed that the recombinant ω-TA has a specific activity of 1.19 U/mg at pH 8.5, 40 °C. The substrate acceptability test showed that ω-TA has significant reactivity to aromatic amino donors and amino receptors. More importantly, the ω-TA also exhibited a good affinity towards some cyclic substrates such as 1-Boc-3-piperidone. The homology model of the ω-TA was built by Discovery Studio and docking was performed to describe the relative activity towards some substrates. The ω-TA was evolved by site-saturation mutagenesis and found that the Q192G mutant increased the activity to the (R)-α-methylbenzylamine (MBA) by around seven-fold. The Q192G mutant was then used to convert two cyclic ketones, N -Boc-3-pyrrolidinone and N -Boc-3-aminopiperidine, and the conversions were both improved compared to the parental ω-TA.
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Figure 2
Figure 3
Figure 4
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Figure 10
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