Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

Guo, Fa-Mou; Wu, Jianping; Yang, Lirong; Xu, Gang

doi:10.1016/j.procbio.2015.05.005

Cited by 14 publications

(4 citation statements)

References 45 publications

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“…Mannanase activities also decreased when temperature increased to 37°C as a result of partially folded protein chains leading to the formation of inclusion bodies. Our results concurred with previous studies of range 18-30°C by EL-Baky et al (2015) and Guo et al (2015). A suitable temperature for E. coli KMAN-3 was different to the camel's HSPA6 nucleotide and amino acid sequences, while the interleukin (IL)-37 protein from mammalian species exhibited high production at an ambient temperature of 37°C (Guo et al, 2015).…”

Section: Discussionsupporting

confidence: 91%

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2021

Int. J. Appl. Microbiol. Biotechnol. Res.

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Most of the recombinant proteins expressed in Escherichia coli were typically employed to isolate proteins from either cytoplasm or periplasm. Optimization of mannanase from the recombinant Escherichia coli KMAN-3 by induction system to obtain extracellular enzyme was presented. Three media as Luria-Bertani (LB), mM9 and mM9NG were studied with enzyme production from mM9 40% cheaper than the others were obtained. Further study on three induction systems of IPTG (IP), lactose (L) and lactose auto-induction (LA) resulted in maximum total mannanase activities of 77.90±1.23, 33.44±0.48 and 61.64±0.01 U/ml, respectively at 18-24 h, correlating with results of transcriptional analysis of the kman-3 gene, except for the LA treatment that presented disparities of its transcription and translation. The lactose auto-induction system showed higher extracellular relative activities of 69.39% while the other two displayed high intracellular activities of 54.16-83.69%. Supplementation of 0.05% (w/v) glucose in mM9 medium gave the highest mannanase production rate of 3.94±0.31 U/ml•h, while induction temperatures of 26 and 30°C showed higher production rates than 20 and 37°C at 1.26 and 3.96 times, respectively. Results suggested the potential of auto-induction using lactose as a cheap inducer to facilitate upscaled major extracellular mannanase production to generate a value-added product such as manno-oligosaccharides in the future.

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Section: Discussionsupporting

confidence: 91%

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2021

Int. J. Appl. Microbiol. Biotechnol. Res.

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“…As expected, prediction using the RBS library calculator (https: //salis.psu.edu/)) [24] showed that elongation by adding an oligopeptide induced a change in the translation initiation rate (TIR). These results suggested that mutagenesis of only terminal regions could effect on the translation rate properties of difficult-to-express proteins [25]. In addition, the resulting bias in the length and sequence of the selected mutants strongly supported the view that histidine-enriched oligopeptide sequences in the N-terminal region of recombinant proteins in E. coli favor a relatively high expression level and solubility, as observed in our previous report [23].…”

Section: Screening and Sequence Analysis Of Recombinant Proteins Withsupporting

confidence: 79%

Recombinant Protein Disulfide Isomerase A3 with an Elongated Peptide Tag Production Process Using Escherichia coli

Lee¹,

Park²

2018

Microbiology and Biotechnology Letters

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Protein disulfide isomerase A3 (PDIA3) is a major member of the protein disulfide isomerase (PDI) family. PDI proteins commonly reside in the endoplasmic reticulum and mediate important thiol-disulfide interchanges during post-translational protein folding. Unlike other PDI family members, PDIA3 is ubiquitous in various organ systems. However, its physiological activity varies in other tissues. PDIA3 has been associated with cancer, airway inflammation, neurodegenerative diseases, and metabolic diseases. However, the mechanisms of the association of PDIA3 with these pathological conditions remain unclear. Recombinant PDIA3 (rPDIA3) is needed to clarify the interactions between PDIA3 and certain physiological phenomena. In the present study, we aimed to produce highly purified rPDIA3 for use in pathological experiments. We expressed rPDIA3 with a histidine-enriched elongated peptide tag in Escherichia coli and obtained rPDIA3 at 97.8% purity using consecutive His-tag and reverse-phase chromatography. Elongated peptide tags screened from artificially designated library had dual functions for protein expression and simple purification.

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“…The decreased specific activity might be caused by the His-tag, which was omitted in the published study. Although it is generally assumed that the His-tag has minimal or no effect on the functionality or structure of most of proteins, a negative impact cannot be excluded, as a few studies revealed the influence of the His-tag on the properties such as a decrease in the enzyme’s activity [ 29 , 30 ]. The highest specific activity of the three kinases was determined for the Aj PPK2 with 69.2 U mg −1 .…”

Section: Resultsmentioning

confidence: 99%

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

et al. 2021

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Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2’3’-cGAMP) formation was successfully developed. The 2’3’‑cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2’3’-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2’3’-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.

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Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

Cited by 14 publications

References 45 publications

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Untitled

Recombinant Protein Disulfide Isomerase A3 with an Elongated Peptide Tag Production Process Using Escherichia coli

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

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