Catalytically active inclusion bodies (CatIBs) induced by terminally attached self-assembling coiled-coil domains: To enhance the stability of (R)-hydroxynitrile lyase

Pei, Xiaolin; Wang, Jiapao; Zheng, Haoteng; Xiao, Qinjie; Wang, Anming; Su, Weike

doi:10.1016/j.enzmictec.2021.109915

Cited by 6 publications

(5 citation statements)

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“…This result differs from that of soluble LipAMS8 lipase and lipolytic enzymes of family 1.3 with optimum pH of 10.0 as reported (Rozi et al, 2022),which almost lost their activity at low pH value. It has also been observed that CatIBs usually show strikingly different characteristics toward low pH as it was reported At-HNL-CatIBs retain up to 70% of their activity at low pH (Pei et al, 2022). This ability of CatIBs to withstand low pH can be exploitable, especially in carrying out reactions that require an acidic medium.…”

Section: Effect Of Ph On Activity and Stability Of Lipams8 Catibsmentioning

confidence: 82%

Catalytically Active Inclusion Bodies of Recombinant LipAMS8 lipase from Antarctic Pseudomonas sp

Bello,

Sabri,

Yahaya

et al. 2024

Preprint

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Heterologous expression of some recombinant protein in E. coli. triggered the formation of inclusion bodies. Recent studies revealed that the aggregated proteins formed are folded correctly and retain their native-like structure, hence possessing catalytic activity. LipAMS8 lipase originally isolated from Antarctic Pseudomonas was overexpressed in E. coli. BL21(De3)/pET32b and resulted in the formation of inclusion bodies. The inclusion bodies were isolated using mild solubilising agents, 50 mM Tris-HCl, 50 mM NaCl, and 1% Triton-X 100 (pH 8.0). Scanning electron microscopy (SEM) analysis depicted the particles as rod-like structures ranging from 1 µm to 100 nm. Biochemical characterisation of the inclusion bodies showcased their catalytic activity, optimum temperature, pH stability, metal ion interaction, and high tolerance to organic solvents. Retaining significant residual activity up to 50% over a range of pH and temperature. Storage stability of LipAMS8 CatIBs at 40C and 250C revealed that it could retain more than 50% of its activity for up to thirteen and eight weeks respectively. This indicated the novelty of the inclusion bodies of LipAMS8 lipase, which is expressed naturally and displays outstanding properties of high activity, pH stability, and extreme tolerance to organic solvents. LipAMS8 lipase CatIBs are catalytically active inclusion bodies that occur naturally as the recombinant enzyme in heterologous expression and can influence the production of cold-active lipase which is highly demanded in various industries for their production processes.

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Section: Effect Of Ph On Activity and Stability Of Lipams8 Catibsmentioning

confidence: 82%

Catalytically Active Inclusion Bodies of Recombinant LipAMS8 lipase from Antarctic Pseudomonas sp

Bello,

Sabri,

Yahaya

et al. 2024

Preprint

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“…Also, these two enzymes exhibited high thermal stability making them more promising to synthesize chiral cyanohydrins very efficiently. [17] AtHNL immobilized inside siliceous monolith (AtHNL-MH) flow microreactor was tested for benzaldehyde and its analogs 4-fluorobenzaldehyde, 4-methoxybenzaldehyde and 4-(trifluoromethyl)benzaldehyde in the synthesis of their respective (R)-cyanohydrins. The usage of flow microreactor has suppressed the background reaction and facilitated high substrate conv.…”

Section: Asymmetric Synthesis Using Natural Nucleophile (Cyanide)mentioning

confidence: 99%

“…The pH‐dependent half‐lives of NSPdoT‐ At HNL and HVdoT‐ At HNL drastically increased to 330±12 min at pH 4.5, 80 fold higher than the WT. Also, these two enzymes exhibited high thermal stability making them more promising to synthesize chiral cyanohydrins very efficiently [17] . At HNL immobilized inside siliceous monolith ( At HNL‐MH) flow microreactor was tested for benzaldehyde and its analogs 4‐fluorobenzaldehyde, 4‐methoxybenzaldehyde and 4‐(trifluoromethyl)benzaldehyde in the synthesis of their respective ( R )‐cyanohydrins.…”

Section: Synthetic Scope Of Newly Discovered Hnlsmentioning

confidence: 99%

Hydroxynitrile Lyase Discovery, Engineering, and Promiscuity towards Asymmetric Synthesis: Recent Progress

Priya,

Padhi

2023

Eur J Org Chem

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Hydroxynitrile lyases (HNLs) primarily catalyze the stereoselective synthesis of cyanohydrins, but some of them also carry out asymmetric nitroaldol synthesis (Henry reaction). By virtue of the importance of asymmetric C‐C bond formation, and the wide application of chiral cyanohydrins and β‐nitroalcohols, HNLs have gained significance in organic synthesis. Over the years new HNLs have been discovered and their synthetic scope are evaluated. Engineering of HNLs has contributed towards increasing not only the catalytic toolbox but also broad substrate selectivity, stereoselectivity, enzyme stability and application of the enzymes for different transformations other than the conventional ones. This review describes the recent discoveries of diverse hydroxynitrile lyases, and the developments to date since 2016, with respect to their biocatalytic applications in the asymmetric synthesis using both natural and promiscuous reactions.

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“…To facilitate the immobilization process, many molecular immobilization strategies have been proposed recently. 22−24 In particular, many auto-assembly tags such as 18A, 25 ELK16, 26 L6KD, 27 GFIL8, 28 NSPdoT, HVdoT, and DMdoT 29 have been developed for triggering the self-assembly of enzymes in vivo. Recently, Wang et al reported CipA and CipB from Photorhabdus luminescens as universal scaffolds for organizing enzymes into protein crystalline inclusions (PCIs) by Nterminal fusion.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Enzyme immobilization confines the soluble enzymes on insoluble carriers through physical or chemical methods. , Considering that the catalytic performance of enzymes is also impacted by the properties of carrier materials and the roles of chemical reagents, the laborious screening for suitable carriers or chemical reagents is always indispensable. To facilitate the immobilization process, many molecular immobilization strategies have been proposed recently. − In particular, many auto-assembly tags such as 18A, ELK16, L6KD, GFIL8, NSPdoT, HVdoT, and DMdoT have been developed for triggering the self-assembly of enzymes in vivo . Recently, Wang et al reported CipA and CipB from Photorhabdus luminescens as universal scaffolds for organizing enzymes into protein crystalline inclusions (PCIs) by N-terminal fusion .…”

Section: Introductionmentioning

confidence: 99%

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate

Wang

Zhao

et al. 2023

ACS Synth. Biol.

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3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient autoassembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.

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Catalytically active inclusion bodies (CatIBs) induced by terminally attached self-assembling coiled-coil domains: To enhance the stability of (R)-hydroxynitrile lyase

Cited by 6 publications

References 50 publications

Catalytically Active Inclusion Bodies of Recombinant LipAMS8 lipase from Antarctic Pseudomonas sp

Catalytically Active Inclusion Bodies of Recombinant LipAMS8 lipase from Antarctic Pseudomonas sp

Hydroxynitrile Lyase Discovery, Engineering, and Promiscuity towards Asymmetric Synthesis: Recent Progress

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate

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