Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide

Liu, Yi; Cui, Wenjing; Liu, Zhongmei; Cui, Youtian; Xia, Yuanyuan; Kobayashi, Michihiko; Zhou, Zhemin

doi:10.1016/j.jbiosc.2014.02.015

Cited by 28 publications

(12 citation statements)

References 19 publications

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“…We previously showed that S1 (AEAEAKAK) 2 , a SAP originating from the Zuotin protein sequence, improved the production and thermal stability of the LOX protein when fused to its N-terminus in E. coli [14]. The positive effects of SAPs on enzyme activity or stability were also observed in the SAP fusions of amylase [15] and nitrile hydratase [16]. We then generated an S1 variant (AEAEAHAH) 2 with the ability to benefit the production, thermal stability, and purification yield of recombinant proteins in E. coli [17].…”

Section: Introductionmentioning

confidence: 99%

An efficient expression tag library based on self-assembling amphipathic peptides

Zhao

Liu

et al. 2019

Microb Cell Fact

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Background Self-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited. Results To improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK) 2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1 nv 1 (ANANARAR) 10 . The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), l -asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 – + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively. Conclusions This is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production. Electronic supplementary material The online version of this article (10.1186/s12934-019-1142-9) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

An efficient expression tag library based on self-assembling amphipathic peptides

Zhao

Liu

et al. 2019

Microb Cell Fact

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“…coli harboring H-NHase could be eliminated by augmenting the quantity of biomass through high-density fermentation. In addition, the capacity for product-tolerance of NHase could be enhanced by protein engineering [27]. The best known strategy is to fuse self-assembling peptide to the N-terminus of NHase, which can largely enhance the capacity for tolerance to high-concentration product [27].…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the capacity for product-tolerance of NHase could be enhanced by protein engineering [27]. The best known strategy is to fuse self-assembling peptide to the N-terminus of NHase, which can largely enhance the capacity for tolerance to high-concentration product [27]. This strategy may also elevate the tolerance to product in recombinant E .…”

Section: Resultsmentioning

confidence: 99%

Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1

Yao

Zhang²,

Liu

et al. 2017

PLoS ONE

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Nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 is widely used for industrial production of acrylamide and nicotinamide. However, the two types of NHases (L-NHase and H-NHase) from R. rhodochrous J1 were only slightly expressed in E. coli by routine methods, which limits the comprehensive and systematic characterization of the enzyme properties. We successfully expressed the two types of recombinant NHases in E. coli by codon-optimization, engineering of Ribosome Binding Site (RBS) and spacer sequences. The specific activity of the purified L-NHase and H-NHase were 400 U/mg and 234 U/mg, respectively. The molecular mass of L-NHase and H-NHase was identified to be 94 kDa and 504 kDa, respectively, indicating that the quaternary structure of the two types of NHases was the same as those in R. rhodochrous J1. H-NHase exhibited higher substrate and product tolerance than L-NHase. Moreover, higher activity and shorter culture time were achieved in recombinant E. coli, and the whole cell catalyst of recombinant E. coli harboring H-NHase has equivalent efficiency in tolerance to the high-concentration product relative to that in R. rhodochrous J1. These results indicate that biotransformation of nitrile by R. rhodochrous J1 represents a potential alternative to NHase-producing E. coli.

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“…According to our previous work [ 3 , 5 , 7 , 8 , 19 – 21 ] and others’ reports [ 4 , 13 , 22 ] about the NHase activators, the P14K is necessary for the functional expression of NHase in B. pallidus RAPc8, C. testosteroni 5-MGAM-4D and P. putida NRRL-18668. Since the amino acid sequence of NHase in B. petrii possessed a high similarity (more than 90 %) with that in P. putida NRRL-18668, it is proposed that the P14K in B. petrii is also necessary for NHase activation.…”

Section: Resultsmentioning

confidence: 99%

Successful expression of the Bordetella petrii nitrile hydratase activator P14K and the unnecessary role of Ser115

et al. 2016

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BackgroundThe activator P14K is necessary for the activation of nitrile hydratase (NHase). However, it is hard to be expressed heterogeneously. Although an N-terminal strep tagged P14K could be successfully expressed from Pseudomonas putida, various strategies for the over-expression of P14K are needed to facilitate further application of NHase.ResultsP14K was successfully expressed through fusing a his tag (his-P14K), and was over-expressed through fusing a gst tag (gst-P14K) at its N-terminus in the NHase of Bordetella petrii DSM 12804. The stability of gst-P14K was demonstrated to be higher than that of the his-P14K. In addition, the Ser115 in the characteristic motif CXLC-Ser115-C of the active center of NHase was found to be unnecessary for NHase maturation.ConclusionsOur results are not only useful for the NHase activator expression and the understanding of the role of Ser115 during NHase activation, but also helpful for other proteins with difficulty in heterologous expression.

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Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide

Cited by 28 publications

References 19 publications

An efficient expression tag library based on self-assembling amphipathic peptides

An efficient expression tag library based on self-assembling amphipathic peptides

Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1

Successful expression of the Bordetella petrii nitrile hydratase activator P14K and the unnecessary role of Ser115

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