Quanfu Wang

Molecular cloning, expression and enzymatic characterization of glutathione S-transferase from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506

A glutathione S-transferase (GST) gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506 (namely PsGST), was cloned and expressed in Escherichia coli. The open reading frame of PsGST comprised 654 bp encoding a protein of 217 amino acids with a calculated molecular size of 24.3 kDa. The rPsGST possesses the conserved amino acid defining the binding sites of glutathione (G-site) and substrate binding pocket (H-site) in GST N_3 family. PsGST was expressed in E. coli and the recombinant PsGST (rPsGST) was purified by Ni-affinity chromatography with a high specific activity of 74.21 U/mg. The purified rPsGST showed maximum activity at 40 °C and exhibited 14.2% activity at 0 °C. It was completely inactivated at 50 °C for 40 min. These results indicated that rPsGST was a typical cold active GST with low thermostability. The enzyme was little affected by H2O2 and Triton X-100, and 50.2% of the remaining activity was detected in the presence of high salt concentrations (2M NaCl). The enzymatic Km values for CDNB and GSH was 0.22 mM and 1.01 mM, respectively. These specific enzyme properties may be related to the survival environment of Antarctic sea ice bacteria.

Cloning, expression and biochemical characterization of recombinant superoxide dismutase from Antarctic psychrophilic bacterium Pseudoalteromonas sp. ANT506.

In this study, a superoxide dismutase gene (PsSOD) from Pseudoalteromonas sp. ANT506 was cloned and over expressed in Escherichia coli. The PsSOD has an open reading frame of 582 bp with a putative product of 193 amino acid residue and an estimated molecular size of 21.4 kDa. His‐tagged PsSOD was subsequently purified 12.6‐fold by Ni‐affinity chromatography and the yield of 22.9%. The characterization of the purified rPsSOD exhibited maximum activity at 30 °C and pH 8.0. The enzyme exhibited 13.9% activity at 0 °C and had high‐thermo lability at higher than 50 °C. rPsSOD exhibited well capability to 2.5 M NaCl (62.4%). These results indicated that rPsSOD exhibited special catalytic properties.

Cloning, Expression, Purification, and Characterization of Glutaredoxin from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. AN178

Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Highly sensitive and selective fluorescence detection of Hg(II) ions based on R-phycoerythrin from Porphyra yezoensis

R-Phycoerythrin (R-PE) is a kind of natural fluorescent protein from marine Porphyra yezoensis. A highly sensitive and selective fluorimetric method has been developed to detect Hg2+ ions utilizing R-PE as a fluorescent probe. R-PE could respond selectively to Hg2+ ions among a series of metal ions. The reaction mechanism was investigated through UV-vis, fluorescence microscope imaging and fluorescence measurements. It was demonstrated that the interaction between the R-PE fluorescent probe and Hg2+ ions would cause fluorescence quenching. Besides, there was a linear relationship between R-PE fluorescence intensities and Hg2+ concentrations. A novel method was thus explored to detect Hg2+ ions with high sensitivity, selectivity, and a broad linear range of 0.0010–25.0 μM, which could allow for Hg2+ ions down to 0.0130 μM with a relative standard deviation of about 1.60%. Additionally, the method was successfully applied to detect Hg2+ ions in diverse water samples, showing the appropriate rates of recovery between 92.0% and 108.0%. This highly sensitive and selective natural green fluorescent probe of R-PE should have huge potential applications for detecting Hg2+ ions in diverse environments.

Crosslinking catalysis-active center of hemin on the protein scaffold toward peroxidase mimic with powerful catalysis

An enzyme mimic synthesis protocol has been proposed by simply cross-linking the redox active center of peroxidase onto a protein scaffold. Colorimetric assays and kinetic studies indicate that the developed peroxidase mimic can present much stronger catalysis and better aqueous stability than native hemin.

A glutathione peroxidase from Antarctic psychrotrophic bacterium Pseudoalteromonas sp. ANT506: Cloning and heterologous expression of the gene and characterization of recombinant enzyme

ABSTRACT A glutathione peroxidase (GPx) gene, designated as PsGPx, was cloned from Antarctic psychrotrophic bacterium Pseudoalteromonas sp. ANT506 and expressed in Escherichia coli. The full-length PsGPx contained a 585-bp encoding 194 amino acids with predicted molecular masses of approx. 21.7 kDa. Multiple sequence alignments revealed that PsGPx belonged to the thioredoxin-like superfamily. PsGPx was heterologously overexpressed in E. coli, purified and characterized. The maximum catalytic temperature and pH value for recombinant PsGPx (rPsGPx) were 30°C and pH 9.0, respectively. rPsGPx retained 45% of the maximum activity at 0°C and exhibited high thermolability with a half-life of approx. 40 min at 40°C. In addition, the enzymatic activity of rPsGPx was still manifested under 3 M NaCl. The Km and Vmax values of the recombinant enzyme using GSH and H2O2 as substrates were 1.73 mM and 16.28 nmol/mL/min versus 2.46 mM and 21.50 nmol/mL/min, respectively.

attB site disruption in marine Actinomyces sp. M048 via DNA transformation of a site-specific integration vector.

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A φC31‐derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi‐parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38±0.41)×10−5 exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tü284). HPLC–MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A–C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD+ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

Cloning, expression and enzymatic characteristics of a 2-Cys peroxiredoxin from Antarctic sea-ice bacterium Psychrobacter sp. ANT206

Peroxiredoxin (Prx, EC 1.11.1.15) is a family of the thiol-dependent antioxidant enzyme. In this study, a cold-adapted Prx gene from Antarctic psychrophilic bacterium Psychrobacter sp. ANT206 (PsPrx) consisted of an open reading frame (ORF) of 567 bp was cloned. Amino acid sequence analysis revealed that PsPrx contained one catalytic site (Thr45, Cys48 and Arg121) and could be categorized as a typical 2-Cys Prx. Compared with the mesophilic StPrx, PsPrx with a reduced amount of hydrogen bonds and salt bridges and other characteristics, may be responsible for its enzymatic stability and flexibility at low temperature. The recombinant PsPrx (rPsPrx) was purified to homogeneity by Ni-NTA and its enzymatic characterization was described. Interestingly, rPsPrx exhibited the maximum activity at 30 °C and remained 42.6% of its maximum activity at 0 °C. rPsPrx was a salt-tolerance enzyme that showed 42.2% of its maximum activity under 2.5 M NaCl. The kinetic parameters of different substrates revealed that it could efficiently catalyze the peroxides, especially H2O2 and t-BOOH (tert‑butyl hydroperoxide). Moreover, rPsPrx exhibited the ability to protect super-coiled DNA from oxidative damage. These results indicated that rPsPrx has special catalytic properties and may be a promising candidate for food and industrial applications.

Predictive modeling of surimi cake shelf life at different storage temperatures

The Arrhenius model of the shelf life prediction which based on the TBARS index was established in this study. The results showed that the significant changed of AV, POV, COV and TBARS with temperature increased, and the reaction rate constants k was obtained by the first order reaction kinetics model. Then the secondary model fitting was based on the Arrhenius equation. There was the optimal fitting accuracy of TBARS in the first and the secondary model fitting (R2≥0.95). The verification test indicated that the relative error between the shelf life model prediction value and actual value was within ±10%, suggesting the model could predict the shelf life of surimi cake.