Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System
Compared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restr...
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2021-09-01
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doaj-2e7c62b4fecb488c85b6efcb43c76f5b2021-09-23T05:02:42ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852021-09-01910.3389/fbioe.2021.747404747404Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration SystemWei Luo0Jinglong Xu1Huiying Chen2Huili Zhang3Peilong Yang4Xiaobin Yu5The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaCollege of Chemistry and Bioengineering, Guilin University of Technology, Guilin, ChinaCollege of Life Sciences, University of Shihezi, Shihezi, ChinaKey Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Institute of Feed Research of CAAS, Beijing, ChinaThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaCompared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restricts the large-scale preparation of L-Asn by biocatalysis. In this study, gene mining strategy was used to search for novel AS with high enzyme activity by expressing them in Escherichia coli BL21 (DE3) or Bacillus subtilis WB600. The obtained LsaAS-A was determined for its enzymatic properties and used for subsequent preparation of L-Asn. In order to reduce the use of ATP, a class III polyphosphate kinase 2 from Deinococcus ficus (DfiPPK2-Ⅲ) was cloned and expressed in E. coli BL21 (DE3), Rosetta (DE3) or RosettagamiB (DE3) for ATP regeneration. A coupling reaction system including whole cells expressing LsaAS-A and DfiPPK2-Ⅲ was constructed to prepare L-Asn from L-aspartic acid (L-Asp). Batch catalytic experiments showed that sodium hexametaphosphate (>60 mmol L−1) and L-Asp (>100 mmol L−1) could inhibit the synthesis of L-Asn. Under fed-batch mode, L-Asn yield reached 90.15% with twice feeding of sodium hexametaphosphate. A final concentration of 218.26 mmol L−1 L-Asn with a yield of 64.19% was obtained when L-Asp and sodium hexametaphosphate were fed simultaneously.https://www.frontiersin.org/articles/10.3389/fbioe.2021.747404/fulll-Asparagineasparagine synthaseclass III polyphosphate kinaseATP regeneration systembiocatalysis and biotransformation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Wei Luo Jinglong Xu Huiying Chen Huili Zhang Peilong Yang Xiaobin Yu |
spellingShingle |
Wei Luo Jinglong Xu Huiying Chen Huili Zhang Peilong Yang Xiaobin Yu Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System Frontiers in Bioengineering and Biotechnology l-Asparagine asparagine synthase class III polyphosphate kinase ATP regeneration system biocatalysis and biotransformation |
author_facet |
Wei Luo Jinglong Xu Huiying Chen Huili Zhang Peilong Yang Xiaobin Yu |
author_sort |
Wei Luo |
title |
Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System |
title_short |
Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System |
title_full |
Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System |
title_fullStr |
Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System |
title_full_unstemmed |
Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System |
title_sort |
synthesis of l-asparagine catalyzed by a novel asparagine synthase coupled with an atp regeneration system |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Bioengineering and Biotechnology |
issn |
2296-4185 |
publishDate |
2021-09-01 |
description |
Compared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restricts the large-scale preparation of L-Asn by biocatalysis. In this study, gene mining strategy was used to search for novel AS with high enzyme activity by expressing them in Escherichia coli BL21 (DE3) or Bacillus subtilis WB600. The obtained LsaAS-A was determined for its enzymatic properties and used for subsequent preparation of L-Asn. In order to reduce the use of ATP, a class III polyphosphate kinase 2 from Deinococcus ficus (DfiPPK2-Ⅲ) was cloned and expressed in E. coli BL21 (DE3), Rosetta (DE3) or RosettagamiB (DE3) for ATP regeneration. A coupling reaction system including whole cells expressing LsaAS-A and DfiPPK2-Ⅲ was constructed to prepare L-Asn from L-aspartic acid (L-Asp). Batch catalytic experiments showed that sodium hexametaphosphate (>60 mmol L−1) and L-Asp (>100 mmol L−1) could inhibit the synthesis of L-Asn. Under fed-batch mode, L-Asn yield reached 90.15% with twice feeding of sodium hexametaphosphate. A final concentration of 218.26 mmol L−1 L-Asn with a yield of 64.19% was obtained when L-Asp and sodium hexametaphosphate were fed simultaneously. |
topic |
l-Asparagine asparagine synthase class III polyphosphate kinase ATP regeneration system biocatalysis and biotransformation |
url |
https://www.frontiersin.org/articles/10.3389/fbioe.2021.747404/full |
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