Expression of enzymes for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis and their preparation for PAPS synthesis and regeneration.

The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.

High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin.

AIMS: One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large-scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. METHODS AND RESULTS: The 6-O-sulfotransferase isoform-3 (6-OST-3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria-Bertani medium in shake flasks and inducing with isopropyl ß-D-1-thiogalactopyranoside at an optical density of 0·6-0·8. The production of larger amounts of 6-OST-3 required fed-batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5-20 mg g-cell-dry weight(-1)) of active 6-OST-3 with excellent productivity (2-5 mg l(-1) h(-1)). CONCLUSIONS: The production of 6-OST-3 in a fed-batch fermentation on an inexpensive carbon source has been demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to scale-up the production of heparin biosynthetic enzymes, such as 6-OST-3, is critical for scaling-up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal-sourced anticoagulant product currently on the market.

Light-activated porphyrin-based formulations to inactivate bacterial spores.

AIM: The objective of this study was to develop porphyrin-based formulations to inactivate Bacillus spores. We probed the effect of porphyrins alone and in combination with germinants against both Bacillus cereus and Bacillus anthracis spores in the presence of light. METHODS AND RESULTS: We tested the effect of two different porphyrins, amine-modified protoporphyrin IX (PPIX) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Treatment with the porphyrins alone did not significantly influence spore viability. However, when spores were pretreated with a solution containing the germinants, l-alanine and inosine, the spore viability dropped by as much as 4.5 logs in the presence of light. The extent of inactivation depended on the germination conditions and the type of porphyrin used, with TMP being more effective. CONCLUSION: Porphyrins can be used effectively in combination with germinants to inactivate Bacillus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide evidence that porphyrins can be used to inactivate Bacillus spores in the presence of germinants and light irradiation. This finding may be general and may be extended to spores of other pathogens.

Selective assembly of multi-component nanosprings and nanorods.

This communication proposes a new approach to create complex hierarchical nano-to-meso-scale architectures based on the use of biological connector molecules to direct the assembly of uniquely shaped multi-component nanostructures fabricated using glancing angle deposition (GLAD). Multiple sets of 50-nm-wide and 150 to 650-nm-tall Si-Cr/Au multi-stack zigzag nanosprings and nanorods are grown by GLAD on Si substrates. Nanorods, chosen for selective assembly, are detached from the substrate, suspended in an aqueous solution, and their surfaces are selectively functionalized by attaching biotin and streptavidin connector-molecules to the Au-regions. Successive mixing of different suspensions leads to the end-to-end assembly of long and short nanorods. This technique provides the path to build hybrid nano-architectures including nano-honeycombs, nanoladders, and 3D nanorod networks, comprised of controlled material combinations.

Biocatalytic plastics as active and stable materials for biotransformations.

Enzyme-containing polymeric materials have been developed that have high activity and stability in both aqueous and organic media. These biocatalytic plastics, containing alpha-chymotrypsin and subtilisin Carlsberg, can contain up to 50% (w/w) total protein in plastic materials such as poly(methyl methacrylate, styrene, vinyl acetate, and ethyl vinyl ether). The activation achieved in organic solvents by incorporating proteases in plastic matrices allows for the efficient synthesis of peptides, and sugar and nucleoside esters. The marriage of enzyme technology with polymer chemistry opens up an array of unique applications for plastic enzymes, including active and stable biocatalysts in paints, coatings, resins, foams, and beads, as well as membranes, fibers, and tubings.

The evolution of biotransformation technologies.

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.

Non-aqueous enzymology.

Compelling evidence has been obtained during the past year that enzymes retain their native active-site structure in organic solvents, and yet the properties of the solvent significantly affect enzyme kinetics. Fundamental advances in enzymatic catalysis in monophasic organic media are discussed and selected applications in the areas of asymmetric, polymer and chemoenzymatic syntheses are highlighted.

Enzymology in monophasic organic media.

Over the past year, an important area of research has been directed towards the fundamental aspects of enzymes and new applications of enzymology in monophasic organic media. Much of this research has focused on the factors that influence enzymatic catalysis in monophasic organic solvents, including the importance of enzyme-associated water, and the effect of organic solvents on enzyme structure and thermodynamic features. From an applications perspective, new advances in the use of enzymes in organic and polymer syntheses and optical resolutions have been made.

Enzymatic and chemoenzymatic approaches to polymer synthesis.

The function of many specialized polymers calls for properties such as chirality and biodegradability. The stereo, -positional-and chemo-selectivities characteristic of enzymatic catalysis are highly desirable attributes for incorporation into strategies for synthesizing such polymers. Enzymes alone, or in combination with chemical synthesis (i.e. chemoenzymatic methodologies), are finding increased use in the synthesis of novel materials. Potential applications include water-absorbents, hydrogels, biodegradable materials, chiral adsorbents, liquid crystals and permselective membranes.

Combinatorial biocatalysis: a natural approach to drug discovery.

Nature's most potent molecules are produced by enzyme-catalysed reactions, coupled with the natural selection of those products that possess optimal biological activity. Combinatorial biocatalysis harnesses the natural diversity of enzymatic reactions for the iterative synthesis of organic libraries. Iterative reactions can be performed using isolated enzymes or whole cells, in natural and unnatural environments, and on substrates in solution or on a solid phase. Combinatorial biocatalysis is a powerful addition to the expanding array of combinatorial methods for the generation and optimization of lead compounds in drug discovery and development.

Macroporous poly(sucrose acrylate) hydrogel for controlled release of macromolecules.

We have studied the controlled release of proteins from poly(sucrose acrylate) hydrogels. The hydrogels were prepared by a two-step procedure in which sucrose was first acylated to sucrose-1'-acrylate followed by free radical polymerization. By adjusting the cross-link ratio and initial monomer concentration, the swelling ratio of the hydrogel was varied from five to 28. The mechanical strength of these hydrogels was comparable to that of the hydrogels with approximately the same swelling ratio. Scanning electron micrographs and mesh size calculations indicate that the hydrogel is macroporous, suggesting it may be suitable for a variety of biomedical applications. The release kinetics of beta-lactoglobulin, bovine serum albumin and gamma-globulin were studied as a function of initial monomer concentrations for the sucrose-based hydrogel. All of the release profiles were characterized by an initial burst of protein in the first 25 h followed by a long period of sustained release (> 500 h). The magnitude of the initial burst was reduced by increasing the initial monomer concentration and by increasing the molecular weight of the protein. A quantitative model based on the heterogeneous nature of hydrogel was developed to explain the observed release kinetics.

Highly swelling hydrogels from ordered galactose-based polyacrylates.

High swelling galactose-based hydrogels have been prepared using a chemoenzymatic procedure. Regioselective acylation of beta-O-methyl-galactopyranoside in nearly anhydrous pyridine with lipase from Pseudomonas cepacia yields the 6-acryloyl derivative (Compound I). Further lipase-catalysed acylation of the monoacrylate derivative in nearly anhydrous acetone yielded 2,6-diacryloyl-beta-O-methyl galactopyranoside (Compound II) that can act as a cross-linker with a structure similar to that of the sugar-based monomer. The high selectivity of enzyme catalysis yielded apparently highly regular hydrogel networks with swelling ratios at equilibrium ranging from 170 to 1100. elastic moduli ranging from 0.005 to 0.088 MPa and calculated mesh sizes ranging from 1160 to 6600 A. These values are far higher than conventional uncharged or lightly charged hydrogels at similar elastic moduli. Gel swelling was fast, with 75% of the equilibrium swelling value reached in a fractional time of 0.17. Non-selective chemical acryloylation of beta-O-methyl galactopyranoside followed by polymerization yielded a far lower-swelling hydrogel than that obtained using selective enzyme catalysis. These results indicate that the highly regular polymer structure achieved by regioselective enzyme-catalysed acylation yields relatively strong and highly swellable materials. Sugar-based hydrogels, such as those described herein, may find particular use as biomaterials because of their high water content, homogeneity, stability and expected non-toxicity. A wide range of pore sizes can be attained, suggesting that they may also be especially useful as matrices for enzyme immobilization and controlled delivery of biological macromolecules.

Designing enzymes for use in organic solvents.

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.

Purification of glycoproteins by selective transport using concanavalin-mediated reverse micellar extraction.

A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.

Affinity-based reverse micellar extraction and separation (ARMES): a facile technique for the purification of peroxidase from soybean hulls.

A new technique for the purification of proteins has been developed which combines the high selectivity of affinity interaction with the scalability and ease of operation of liquid-liquid extraction. The approach is called affinity-based reverse micellar extraction and separation (ARMES). The salient features of ARMES include the following: (1) intraphasic interaction between the ligand and ligate which provides for high ligand utilization; (2) no chemical modification of the ligand is needed; and (3) ease of operation and inherent scalability due to the use of liquid-liquid extraction. This technique has been used to purify the peroxidase from soybean hulls using the lectin concanavalin A (con A) as a sugar-binding affinity ligand. A purification factor of 30 is achieved to provide a nearly pure peroxidase solution (as determined by HPLC and SDS-PAGE) with nearly complete regeneration of the con A ligand. We propose that ARMES will be useful in the facile purification of complex biomolecules such as glycoform protein variants using lectins as affinity ligands and proteins of therapeutic importance using antibodies as affinity ligands.

Enzymatically and combinatorially generated array-based polyphenol metal ion sensor.

Phenolic polymers were synthesized via soybean hull peroxidase catalysis and used as metal-based sensor components in a polymer array. A sensor array for Fe(3+), Cu(2+), Co(2+), and Ni(2+) has been developed consisting of 15 phenolic homopolymers and copolymers generated from five phenolic monomers by peroxidase-catalyzed oxidative polymerization. Sensing was based on the change of intrinsic polyphenol fluorescence upon addition of a metal ion or a metal ion mixture to an aqueous suspension of a polyphenol. Importantly, the fluorescence response of copolymers differed, in some cases dramatically, from the constituent homopolymers and was dependent upon the relative ratio of monomers that comprise the polymer. This finding suggests that an extremely broad range of sensor arrays can be generated from a limited number of phenolic monomers. Using a statistical analysis, histograms constructed for the four different metal ions yielded unique fingerprints of the array response and can be used to identify specific metal ions.

Parameters affecting the efficiency of affinity-based reversed micellar extraction and separation (ARMES) in glycoprotein purification.

Affinity-based reversed micellar extraction and separation (ARMES) is an effective method for purifying both low and high molecular weight glycoproteins via liquid-liquid extraction. A range of extraction conditions were examined to gain insight into the mechanism of ARMES. Concanavalin A (Con A) was used as the model affinity ligand to bind soybean peroxidase (SBP) and beta-galactosidase as model glycoproteins. Factorial design was used to investigate the effect of various system variables on the extraction of SBP via ARMES. A quadratic model described the systems well, resulting in a standard deviation of 7% between calculated and experimental extraction efficiencies. Sensitivity analysis suggested that the key criteria in ARMES were the NaCl concentration and pH of the aqueous feed phase. Extraction of both glycoproteins decreased above pH 7 but fell to zero only at pH values significantly above the pI of the model glycoproteins and the Con A affinity ligand. It is proposed that the complex of the affinity lectin with the glycoprotein results in a sufficiently hydrophobic species that can be extracted into a reversed micellar organic phase even at pH's far above the pI's of the individual proteins that comprise the complex. This finding has practical considerations for the use of ARMES in the resolution and purification of protein glycoforms.

Catalytic properties and potential of an extracellular protease from an extreme halophile.

An extracellular protease has been isolated and partially purified from the extreme halophile Halobacterium halobium (ATCC 43214). The major enzyme component has a M(r) of 66,000 and is highly dependent upon salt concentrations near saturation for catalytic activity and stability. In aqueous solutions, a decrease in the NaCl concentration from 4 to 1 M results in an increase of nearly three orders of magnitude in the first-order rate constant of inactivation at 30 degrees C. Salt effects the stability of the enzyme in a cooperative manner, with a Hill coefficient of 4.1, which is similar to that of other enzymes from extreme halophiles. The enzyme activity is dramatically affected by the salt concentration, with a loss of 2.5 orders of magnitude in kcat/Km in going from 4 to 0 M NaCl. This loss in catalytic efficiency is primarily due to a dramatic increase in the Km for the substrate in low-salt media. Thermodynamic analysis revealed that this Km increase was mainly the result of increased solubility of the synthetic peptide substrate in low-salt media, which dramatically increases the ground-state stability of the substrate. This results in an effectively reduced substrate partitioning from the bulk solution into the enzyme's active site and an increased value of Km. The halophilic protease is also active in DMF/water mixtures, albeit with novel catalytic properties. In 33% (v/v) DMF in aqueous buffer, the esterase activity of the enzyme is ca. 80-fold higher than the corresponding amidase activity. This contrasts to the situation in pure aqueous buffer, in which the esterase activity is only fourfold higher than the amidase activity. The increased esterase activity relative to amidase activity prompted us to investigate the use of the protease in kinetically controlled peptide synthesis. The enzyme has a broad acyl donor substrate specificity and can effectively use amino acid esters of Phe, Tyr, Trp, Ser, Gly, and Ala. The enzyme is significantly more selective for the amino acid amide, preferring Gly in the P'1 site. A series of glycine-containing oligopeptides have been prepared in yields up to 76% without degradation due to secondary hydrolysis.

Affinity-based separations and purifications. Patents and literature.

The separation and purification of biologically functional molecules (e.g., proteins, antibodies, peptides, hormones, low molecular weight biologicals) is of fundamental importance to biotechnology. Affinity separations have become a particularly attractive method for bioseparations due to their high degree of selectivity. Numerous affinity ligands have been prepared in recent years including lectins, nucleic acids, inhibitors, and immunoresponse agents. Furthermore, a variety of novel supports have been synthesized to aid in the development of commercially useful affinity separation systems. Recent US patents and scientific literature on affinity separations and purifications are surveyed. Patent abstracts are summarized individually and a list of literature references are given.

Selective biotransformations. Patents and literature.

Stereo- and regioselective biotransformations is an area of intense research interest. The ability to take advantage of microorganisms and isolated enzyme systems to perform selective organic syntheses is well known. In recent years, several new methodologies have caused a rapid growth in the area of selective biotransformations. These novel techniques include genetic and protein engineering, biocatalysis in organic media, improved screening procedures, as well as an increased awareness of the value of biotechnology to solve important problems in organic synthesis. Recent US patents and scientific literature on stereo- and regioselective biotransformations are surveyed. Patent abstracts are summarized individually and a list of literature references are given.