Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

Surface display of highly-stable Desulfovibrio vulgaris carbonic anhydrase on polyester beads for CO2 capture.

OBJECTIVES: To engineer a recombinant Escherichia coli to produce polyhydroxyalkanoate biopolymer beads displaying carbonic anhydrase (CA) from Desulfovibrio vulgaris str. "Miyazaki F" (DvCA). RESULTS: The highest measured specific activity of the immobilised CA was 211 U/mg DvCA. The immobilised CA was thermostable, retaining 114 U/mg DvCA of activity after incubation at 90 °C for 1 h. Additionally, the immobilised CA tolerated 30 min incubations in a variety of pH conditions and was especially tolerant of alkaline conditions, retaining 131 U/mg DvCA of activity after pH 12 incubation. CONCLUSION: CA has a potential role in atmospheric CO2 mitigation strategies and the stability of the functionalised beads indicates suitability for use in industrial settings such as coal-fired power plants.

In vivo self-assembly of stable green fluorescent protein fusion particles and their uses in enzyme immobilization.

Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase from Bacillus licheniformis, N-acetyl-d-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to N-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

Polyhydroyxalkanoate synthase fusions as a strategy for oriented enzyme immobilisation.

Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

Bioengineering of bacterial polymer inclusions catalyzing the synthesis of N-acetylneuraminic acid.

N-Acetylneuraminic acid is produced by alkaline epimerization of N-acetylglucosamine to N-acetylmannosamine and then subsequent condensation with pyruvate catalyzed by free N-acetylneuraminic acid aldolase. The high-alkaline conditions of this process result in the degradation of reactants and products, while the purification of free enzymes to be used for the synthesis reaction is a costly process. The use of N-acetylglucosamine 2-epimerase has been seen as an alternative to the alkaline epimerization process. In this study, these two enzymes involved in N-acetylneuraminic acid production were immobilized to biopolyester beads in vivo in a one-step, cost-efficient process of production and isolation. Beads with epimerase-only, aldolase-only, and combined epimerase/aldolase activity were recombinantly produced in Escherichia coli. The enzymatic activities were 32 U, 590 U, and 2.2 U/420 U per gram dry bead weight, respectively. Individual beads could convert 18% and 77% of initial GlcNAc and ManNAc, respectively, at high substrate concentrations and near-neutral pH, demonstrating the application of this biobead technology to fine-chemical synthesis. Beads establishing the entire N-acetylneuraminic acid synthesis pathway were able to convert up to 22% of the initial N-acetylglucosamine after a 50-h reaction time into N-acetylneuraminic acid.

Polyhydroxybutyrate (PHB) produced from red grape pomace: Effect of purification processes on structural, thermal and antioxidant properties.

Red grape pomace was used as a source for poly(3-hydroxybutyrate) (PHB) production, which was then subject to a range of purification processes. The different PHB biopolymers were characterized for chemical structure, crystallinity, thermal properties, colour, release of compounds into different food simulants and antioxidant inhibition, and comparisons were made with a commercially available PHB. An increase in purification steps did not have a significant effect on the high thermal stability of the extracted biopolymer, but it decreased the degree of crystallinity and the presence of amino acids and aromatic compounds. With additional purification, the PHB powders also whitened and the number of components released from the biopolymer into food simulants decreased. The released compounds presented antioxidant inhibition, which has not been previously reported in the literature or with commercially available polyhydroxyalkanoates. This is of great interest for food packaging and biomedical industries where the addition of antioxidant additives to improve PHB functional properties may not be necessary and could be avoided.

The yeast IRC7 gene encodes a ß-lyase responsible for production of the varietal thiol 4-mercapto-4-methylpentan-2-one in wine.

Three varietal thiols are key aroma compounds in Sauvignon Blanc wines: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). Screening of Saccharomyces cerevisiae strains identified a clinical isolate with elevated 4MMP production after fermentation. Bulked Segregant Analysis of a cross between this isolate and the laboratory strain revealed a single major locus for 4MMP production near the telomere of chromosome 6. Deletion of the IRC7 gene from this region in YJM450 reduced 4MMP production below detectable levels, but did not affect yields of 3MH, in Sauvignon Blanc wine. Sequencing revealed that the IRC7 gene in YJM450 had been introgressed from a strain of Saccharomyces paradoxus. Most strains of S. cerevisiae, including the laboratory strain S288C, have a 38-bp deletion that inactivates IRC7. Overexpression of a full-length S. cerevisiae allele of IRC7 in a wine yeast, Zymaflore F15, increased 4MMP production in Sauvignon Blanc wine from undetectable levels (<10 ng L(-1)) to concentrations of 1000 ng L(-1), and also increased 3MH and 3MHA. Biochemical analysis of soluble protein extracts showed that both the cerevisiae and paradoxus IRC7 proteins show ß-lyase activity, with a substrate preference for cys-4MMP over cys-3MH.

Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum, Trichosurus vulpecula.

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum (Trichosurus vulpecula) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.

Escherichia coli NemA is an efficient chromate reductase that can be biologically immobilized to provide a cell free system for remediation of hexavalent chromium.

Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI) to insoluble and relatively non-toxic Cr(III), bacterial bioremediation of Cr(VI) pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI) remediation. To identify novel Cr(VI) reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI) indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI) reductase (k(cat)/K(M)= 1.1×10(5) M(-1) s(-1) with NADH as cofactor). Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI) remediation.