Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio

Du, Kun; Zhao, Jinjin; Sun, Jian; Feng, Wei

doi:10.1021/acs.bioconjchem.7b00043

Cited by 19 publications

(18 citation statements)

References 51 publications

(77 reference statements)

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“…The apparent K m value of 12.02 mM f-LaMOF@DAAO is smaller than the K m value of 15.37 mM free DAAO. 41 It is indicated that f-LaMOF@DAAO exhibited an enhanced affinity toward the substrate. The catalysis efficiency can be measured by the k cat / K m ratio.…”

Section: Results and Discussionmentioning

confidence: 99%

Fabrication of a Fibrous Metal–Organic Framework and Simultaneous Immobilization of Enzymes

et al. 2020

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A nanorod-like lanthanum metal–organic framework (LaMOF) was synthesized in aqueous solution by coordinating La(III) to the ligand 1,3,5-benzenetricarboxylic acid. The fibrous LaMOF was fabricated by splitting the nanorod-like LaMOF in a solution of d -amino acid oxidase, and the enzyme was immobilized simultaneously. Based on SEM and TEM images, STEM mapping, and spectra of XPS and FTIR, the mechanism of formation of the fibrous LaMOF and the distinct interfacial phenomena have been elucidated. The fabrication of the fibrous LaMOF and simultaneous immobilization of the enzyme were carried out in aqueous solutions at room temperature, without using any organic solvent. It is a clean and time- and energy-effective process. This work presents a distinct and clean methodology for the fabrication of the fibrous MOF. Potentially, the environmentally benign methodology can be extended to immobilize other enzymes.

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Section: Results and Discussionmentioning

confidence: 99%

Fabrication of a Fibrous Metal–Organic Framework and Simultaneous Immobilization of Enzymes

et al. 2020

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“…The expression of enzymes is briefly described as follows [34]. E. coli BL21 harboring the plasmids were grown at 25 • C in Luria-Bertani (LB) medium with 50 µg/mL Amp.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Co-Immobilization of Superoxide Dismutase with Catalase on Soft Microparticles Formed by Self-Assembly of Amphiphilic Poly(Aspartic Acid)

Mao

Wang

et al. 2017

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Through genetic engineering technology, catalase (CAT) and superoxide dismutase (SOD) have been separately fused to an elastin-like polypeptide (ELP). Thus, the enzymes can be purified through phase transition. Hexadecylamine-modified poly(aspartic acid) (HPASP) is able to self-assemble, forming soft microparticles. The HPASP microparticles were used to co-immobilize SOD-ELP and CAT-ELP through amidation reaction. Circular dichroism (CD) confirmed that the secondary structures of the co-immobilized enzymes have been preserved. Fluorescence spectra showed that the co-immobilized enzymes exhibited a higher stability than the free enzymes. Dismutation of superoxide by superoxide dismutase (SOD) generates hydrogen peroxide. By using the co-immobilized enzymes (SOD-ELP/CAT-ELP@HPASP), the generated hydrogen peroxide of SOD-ELP can be decomposed in situ by CAT-ELP. Activity assay results demonstrated that the superoxide anion (•O 2 − ) scavenging ability is 63.15 ± 0.75% for SOD-ELP/CAT-ELP@HPASP.The advantages of the approach of enzyme co-immobilization include the fact that the soft support HPASP itself is a polypeptide in nature, the stability of immobilized enzymes is improved, and a high activity has been achieved. Potentially SOD-ELP/CAT-ELP@HPASP can be applied in the cosmetic industry.

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“…Previously, we have specifically ligated two multimeric enzymes with native peptides through intein-mediated in vivo subunit splicing (Du et al, 2017;Li et al, 2018). Herein, we develop the methodology, by using the coiled-coil association of an arginine-rich leucine zipper motif (Z R ) (Moll et al, 2001) and a glutamic acid-rich leucine zipper motif (Z E ) (Moll et al, 2001) to trigger the intein-mediated in vivo subunit splicing.…”

Section: Introductionmentioning

confidence: 99%

Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs

Chen

et al. 2020

Front. Bioeng. Biotechnol.

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For the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated in vivo subunit splicing. The subunit ligation is triggered by the heterodimerization of an arginine rich leucine zipper motif with a glutamic acid rich leucine zipper motif. The one-by-one ligation of hetrosubunits constructs the pairing enzymes RTA&AlaDH and AlaDH&FDH. The ligation modes were analyzed based on blue native polyacrylamide gel electrophoresis (BN-PAGE). The spectra of circular dichroism (CD), fluorescence, and two-dimensional FTIR provide information on the secondary structures and stability of the pairing enzymes. The enzymesubstrate interaction was analyzed based on microscale thermophoresis analysis. In contrast to the mixed three enzymes RTA + AlaDH + FDH, the ligated enzymes RTA&AlaDH + AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity.

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Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio

Cited by 19 publications

References 51 publications

Fabrication of a Fibrous Metal–Organic Framework and Simultaneous Immobilization of Enzymes

Fabrication of a Fibrous Metal–Organic Framework and Simultaneous Immobilization of Enzymes

Co-Immobilization of Superoxide Dismutase with Catalase on Soft Microparticles Formed by Self-Assembly of Amphiphilic Poly(Aspartic Acid)

Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs

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