Mechanism-Guided Computational Design Drives meso-Diaminopimelate Dehydrogenase to Efficient Synthesis of Aromatic d-amino Acids.

Aromatic d-amino acids (d-AAs) play a pivotal role as important chiral building blocks and key intermediates in fine chemical and drug synthesis. Meso-diaminopimelate dehydrogenase (DAPDH) serves as an excellent biocatalyst in the synthesis of d-AAs and their derivatives. However, its strict substrate specificity and the lack of efficient engineering methods have hindered its widespread application. Therefore, this study aims to elucidate the catalytic mechanism underlying DAPDH from Proteus vulgaris (PvDAPDH) through the examination of its crystallographic structure, computational simulations of potential energies and molecular dynamics simulations, and site-directed mutagenesis. Mechanism-guided computational design showed that the optimal mutant PvDAPDH-M3 increased specific activity and catalytic efficiency (kcat/Km) for aromatic keto acids up to 124-fold and 92.4-fold, respectively, compared to that of the wild type. Additionally, it expanded the substrate scope to 10 aromatic keto acid substrates. Finally, six high-value-added aromatic d-AAs and their derivatives were synthesized using a one-pot three-enzyme cascade reaction, exhibiting a good conversion rate ranging from 32 to 84% and excellent stereoselectivity (enantiomeric excess >99%). These findings provide a potential synthetic pathway for the green industrial production of aromatic d-AAs.

Bioinformatics and experimental studies on the structural roles of a surface-exposed α-helix at the C-terminal domain of Chondroitinase ABC I.

A series of single and double mutants generated on residues of a surfaced-exposed helix at the C-terminal domain of chondroitinase ABC I (cABC I) from proteus vulgaris. M886A, G887E, and their respective double mutant, MA/GE were inspired by the sequence of a similar helix segment in 30S ribosomal protein S1. Additionally, M889I, Q891K, and the corresponding double mutant, MI/QK, were made regarding the sequence of a similar helix in chondroitin lyase from Proteus mirabilis. Circular dichroism spectra in the far-UV region, demonstrate that the ordered structure of wild-type (WT), and double mutants are the same; however, the helicity of the ordered structures in MI/QK is higher than that of the WT enzyme. When compared with the single mutants, the double mutants showed higher activity, and that the activity of MI/QK is higher than that of the WT enzyme. Heat-induced denaturation experiments showed that the stability of the tertiary structure of double mutants at moderate temperatures is higher compared with the WT, and single mutants. It concluded that this helix can be considered as one of the hot spots region that can be more manipulated to obtain improved variants of cABC I.

Biochemical characterization of a surfactant-stable keratinase purified from Proteus vulgaris EMB-14 grown on low-cost feather meal.

OBJECTIVES: The bioaccumulation of keratinous wastes from poultry and dairy industries poses a danger of instability to the biosphere due to resistance to common proteolysis and as such, microbial- and enzyme-mediated biodegradation are discussed. RESULTS: In submerged fermentation medium, Proteus vulgaris EMB-14 utilized and efficiently degraded feather, fur and scales by secreting exogenous keratinase. The keratinase was purified 14-fold as a monomeric 49 kDa by DEAE-Sephadex A-50 anion exchange and Sephadex G-100 size-exclusion chromatography. It exhibited optimum activity at pH 9.0 and 60 °C and was alkaline thermostable (pH 7.0-11.0), retaining 87% of initial activity after 1 h pre-incubation at 60 °C. The Km and Vmax of the keratinase with keratin azure were respectively 0.283 mg/mL and 0.241 U/mL/min. Activity of P. vulgaris keratinase was stimulated by Ca2+, Mg2+, Zn2+, Na+ and maintained in the presence of some denaturing agents, except ß-mercaptoethanol while Cu2+ and Pb2+ showed competitive and non-competitive inhibition with Ki 6.5 mM and 17.5 mM, respectively. CONCLUSION: This purified P. vulgaris keratinase could be surveyed for the biotechnological transformation of bioorganic keratinous wastes into valuable products such as soluble peptides, cosmetics and biodegradable thermoplastics.

Ultrasound-assisted production of biodiesel using engineered methanol tolerant Proteus vulgaris lipase immobilized on functionalized polysulfone beads.

In the present study, Proteus vulgaris lipase (PVL) was engineered using directed evolution to increase methanol tolerance so that it would be more tolerant and efficient for harsh conditions employed in biodiesel synthesis, which is limiting their industrial use. The influence of ultrasound under different experimental conditions on the biodiesel conversion yield using methanolysis of non-edible neem oil was also emphasized. A special attention was also paid to the immobilization of lipase on Polysulfone (PS) beads and comparative studies with industrially used Burkholderia cepacia lipase. The Engineered Proteus vulgaris lipase showed >80% activity after 3 h when incubated in 50% methanol with simultaneous sonication. The lipase retained improved longevity (~70% residual activity) over wild-type PVL over repeated use.

Secretory expression of biologically active chondroitinase ABC I for production of chondroitin sulfate oligosaccharides.

Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of I309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp, all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 ±â€¯0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides.

Designing and construction of novel variants of Chondroitinase ABC I to reduce aggregation rate.

Chondroitinase ABC I (cABC I) can degrade inhibitory molecules for axon regrowth at the site of damage after spinal cord injury (SCI). One of the main problems in the practical application is the possibility of structural changes that lead to the inactivation of the enzyme. In current work, three variants of cABC I was designed and constructed by manipulation of a short helix conformation (Gln678-Leu679-Ser680-Gln681); where Gln residues were converted to Glu. According to the enzyme kinetics studies, the catalytic efficiency of the Q681E and double mutant (Q678E/Q681E) increases in comparison with WT enzyme; while that of Q678E decreases. It was also shown that the rate of the inactivation of the enzyme variants over time is greater in WT and Q678E variants than that of the Q681E and double mutant. Negative values of entropy change of thermal inactivation measurements; demonstrate that inactivation of the WT and Q678E variants are mainly originated from aggregation. These observations can be explained by considering the repulsive electrostatic interaction between enzyme molecules that prevents protein aggregation over time. It is concluded that increasing the solubility of the Q681E and double mutant via favorable interactions of surface-exposed charged residues with dipole momentum of water molecules accompanied by the presence of intermolecular repulsive electrostatic interaction leads to decreasing the rate of aggregation in both long-term storage and heat-induced structural changes.

N∆89 and C∆274 Truncated Enzymes of Chondroitinase ABC I Regain More Imperturbable Microenvironments Around Structural Components in Comparison to their Wild Type.

Immune response stimulation and inactivation of chondroitinase ABC I in physiological condition have been limited its use in various clinical conditions as a bacterial enzyme drug. In the present study, we have investigated some structural and functional features of N∆89, C∆274 and N∆89C∆274; three designed truncated cABC I, in order to clarify the unclear role of two terminal parts of cABC I i.e., the 1-89 and 747-1021 amino acids sequences of the full length enzyme through truncation. As a result, the numbers of potential epitopes, the susceptibility to trypsin digestion, ANS fluorescence spectra, and fluorescence quenching using KI and acrylamide were diminished for N∆89 and C∆274 in comparison to the wild type. Secondary and tertiary structure investigation for N∆89 and C∆274 revealed that the intrinsic fluorescence was increased and Far-UV CD spectra were changed accordingly. Relative to the wild type enzyme, 0.164, 0.195 remaining activity and lack of activity was shown with the zymographic assay for N∆89, C∆274 and N∆89C∆274 variants, respectively. The diminished enzyme activity and structural changes suggested a reorientation of microenvironments interactions including cation-π interactions around structural elements toward lowering regional mobility. Constructing applicable truncated cABC I with improved features could be regarded as a strategy to regain new possible functional advantages over the full length enzyme.

Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains.

Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury.

AIMS: Usually, spinal cord injury (SCI) develops into a glial scar containing extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABC (ChABC), from Proteus vulgaris degrading the glycosaminoglycan (GAG) side chains of CSPGs, offers the opportunity to improve the final outcome of SCI. However, ChABC usage is limited by its thermal instability, requiring protein structure modifications, consecutive injections at the lesion site, or implantation of infusion pumps. METHODS: Aiming at more feasible strategy to preserve ChABC catalytic activity, we assessed various stabilizing agents in different solutions and demonstrated, via a spectrophotometric protocol, that the 2.5 mol/L Sucrose solution best stabilized ChABC as far as 14 days in vitro. RESULTS: ChABC activity was improved in both stabilizing and diluted solutions at +37°C, that is, mimicking their usage in vivo. We also verified the safety of the proposed aqueous sucrose solution in terms of viability/cytotoxicity of mouse neural stem cells (NSCs) in both proliferating and differentiating conditions in vitro. Furthermore, we showed that a single intraspinal treatment with ChABC and sucrose reduced reactive gliosis at the injury site in chronic contusive SCI in rats and slightly enhanced their locomotor recovery. CONCLUSION: Usage of aqueous sucrose solutions may be a feasible strategy, in combination with rehabilitation, to ameliorate ChABC-based treatments to promote the regeneration of central nervous system injuries.

Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides.

Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points. One format of IEF, the capillary isoelectric focusing (cIEF), is an attractive approach in that it can be coupled with mass spectrometry (cIEF-MS) to provide online focusing and detection of complex mixtures. In the past three decades, numerous studies have applied cIEF-MS methods to the analysis of protein and peptide mixtures by positive-ion mode mass spectrometry. However, polysaccharide chemists largely rely on negative-ion mode mass spectrometry for the analysis of highly sulfated GAGs. The current study reports a negative-ion mode cIEF-MS method using an electrokinetically pumped sheath liquid nanospray capillary electrophoresis-mass spectrometry (CE-MS) coupling technology. The feasibility of this negative-ion cIEF-MS method and its potential applications are demonstrated using chondroitin sulfate and heparan sulfate oligosaccharides mixtures.

The crystal structure of Proteus vulgaris tryptophan indole-lyase complexed with oxindolyl-L-alanine: implications for the reaction mechanism.

Tryptophan indole-lyase (TIL) is a bacterial enzyme which catalyzes the reversible formation of indole and ammonium pyruvate from L-tryptophan. Oxindolyl-L-alanine (OIA) is an inhibitor of TIL, with a Ki value of about 5 µM. The crystal structure of the complex of Proteus vulgaris TIL with OIA has now been determined at 2.1 Šresolution. The ligand forms a closed quinonoid complex with the pyridoxal 5'-phosphate (PLP) cofactor. The small domain rotates about 10° to close the active site, bringing His458 into position to donate a hydrogen bond to Asp133, which also accepts a hydrogen bond from the heterocyclic NH of the inhibitor. This brings Phe37 and Phe459 into van der Waals contact with the aromatic ring of OIA. Mutation of the homologous Phe464 in Escherichia coli TIL to Ala results in a 500-fold decrease in kcat/Km for L-tryptophan, with less effect on the reaction of other nonphysiological ß-elimination substrates. Stopped-flow kinetic experiments of F464A TIL show that the mutation has no effect on the formation of quinonoid intermediates. An aminoacrylate intermediate is observed in the reaction of F464A TIL with S-ethyl-L-cysteine and benzimidazole. A model of the L-tryptophan quinonoid complex with PLP in the active site of P. vulgaris TIL shows that there would be a severe clash of Phe459 (∼1.5 Šapart) and Phe37 (∼2 Šapart) with the benzene ring of the substrate. It is proposed that this creates distortion of the substrate aromatic ring out of plane and moves the substrate upwards on the reaction coordinate towards the transition state, thus reducing the activation energy and accelerating the enzymatic reaction.

An efficient biocatalytic synthesis of imidazole-4-acetic acid.

OBJECTIVE: To develop a new and efficient biocatalytic synthesis method of imidazole-4-acetic acid (IAA) from L-histidine (L-His). RESULTS: L-His was converted to imidazole-4-pyruvic acid (IPA) by an Escherichia coli whole-cell biocatalyst expressing membrane-bound L-amino acid deaminase (mL-AAD) from Proteus vulgaris firstly. The obtained IPA was subsequently decarboxylated to IAA under the action of H2O2. Under optimum conditions, 34.97 mM IAA can be produced from 50 mM L-His, with a yield of 69.9%. CONCLUSIONS: Compared to the traditional chemical synthesis, this biocatalytic method for IAA production is not only environmentally friendly, but also more cost effective, thus being promising for industrial IAA production.

Improving the stability of chondroitinase ABC I via interaction with gold nanorods.

Chondroitinase ABC I (cABC I) cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is found to prune glycosaminoglycan chains of proteoglycans and facilitate axon regeneration. However, a limiting factor for such application is the enzyme's instability. In this study, the structure and activity of cABC I have been investigated upon interaction with various concentrations of Gold nanorods. The enzyme preserved its major activity with increase in substrate affinity in the presence of the nanostructures. Analysis of circular dichroism spectropolarimetry data showed that secondary structural content of the enzyme slightly increased. The complex form of the enzyme also showed higher storage stability. Fluorescence studies indicated that enzyme obtained more rigidity in its structure. Taking higher stability of enzyme upon interaction, result of this investigation interaction paves the way for utilizing tiny plasmonic nanostructures for fruitful applications in biomedicine.

Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity.

Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.

Investigating the structural and functional features of representative recombinants of chondroitinase ABC I.

Chondroitin Sulfate Proteoglycans (CSPGs) are the main inhibitors for axon regeneration after damaging of Central Nervous System (CNS). Chondroitinase ABC I (cABC I) can degrade CSPGs by removing chondroitin and dermatan sulfate side chains from proteoglycans. Hence, it may be considered as an attractive candidate in biomedicine. For practical applications of this enzyme, increasing the effective circulating level and reducing the number and volume of injections for patients is one of the main concerns which is directly related to conformational stability and catalytic efficiency of the enzyme. Structural examination of C-terminal domain of cABC I reveals that there are a few numbers of residues in helical conformation which are positioned at the context of a cohesive structural organization of ß-strands. In line with our previous studies on C-terminal domain of cABC I and regarding the residues in α-helix conformation; we designed and constructs some representative mutants including M889K, M889L, L679D/M889K and L679S/M889K. According to structural and functional characterization of protein variants and regarding the wide range of variability in determining parameters for ß-sheet conformation, we proposed a model in which the structural integrity of ß-strands at C-terminal domain can be manipulated and directed toward a new patterns of organization, some of them may have positive effects on the structural and functional features of the enzyme. Using this strategy it may be possible to improve functional and structural features of the enzyme by engineering the intra-molecular interactions in positions far from the active site of the enzyme.

Improvement of activity and stability of Chondroitinase ABC I by introducing an aromatic cluster at the surface of protein.

Chondroitinase ABC I (ChABC I) has been shown to depolymerize a variety of glycosaminoglycan substrates and promote regeneration of damaged spinal cord. However, to date, intrathecal delivery methods have been suboptimal largely due to enzyme instability which necessitates repeated administration to the injured loci. Among the aromatic amino acids, tyrosine has been shown to be more effective in creation of stable clusters and further stabilize of the proteins. Bioinformatics approaches have been used to examine the effect of an extra aromatic cluster at the surface of ChABC I. In this study two amino acids i.e., Asn806 and Gln810 were mutated to tyrosine and to alanine as negative control. In this way, four variants i.e., N806Y/Q810Y, N806A/Q810Y, N806Y/Q810A and N806A/Q810A were created. The results showed that N806Y/Q810Y mutation improved both activity and thermal stability of the enzyme while Ala substitution reduced the enzyme activity and destabilized it. Structural analysis of mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of N806Y/Q810Y mutant when compared to the wild type enzyme indicating a more rigid structure of this variant. Moreover, the N806Y/Q810Y enzyme displayed a remarkable resistance against trypsin degradation with a half-life (t1/2) of 45.0min versus 32.5min of wild-type. In conclusion, the data revealed that structural features and activity of ChABC I can be improved by introducing appropriate aromatic clusters at the surface of the enzyme.

Porous silicon nanoparticle as a stabilizing support for chondroitinase.

Chondroitinase ABCI (cABCI) from Proteus vulgaris is a drug enzyme that can be used to treat spinal cord injuries. One of the main problems of chondroitinase ABC1 is its low thermal stability. The objective of the current study was to stabilize the enzyme through entrapment within porous silicon (pSi) nanoparticles. pSi was prepared by an electrochemical etch of p-type silicon using hydrofluoric acid/ethanol. The size of nanoparticles were determined 180nm by dynamic light scattering and the mean pore diameter was in the range of 40-60nm obtained by scanning electron microscopy. Enzymes were immobilized on porouse silicon nanoparticles by entrapment. The capacity of matrix was 35µg enzyme per 1mg of silicon. The immobilized enzyme displayed lower Vmax values compared to the free enzyme, but Km values were the same for both enzymes. Immobilization significantly increased the enzyme stability at various temperatures (-20, 4, 25 and 37°C). For example, at 4°C, the free enzyme (in 10mM imidazole) retained 20% of its activity after 100min, while the immobilized one retained 50% of its initial activity. Nanoparticles loading capacity and the enzyme release rate showed that the selected particles could be a pharmaceutically acceptable carrier for chondroitinase.

Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli.

Chondroitinases (ChSases) are a family of polysaccharide lyases that can depolymerize high molecular weight chondroitin sulfate (CS) and dermatan sulfate (DS). In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in different cells like normal cells and cancer cells and the expression is relatively insensitive to experimental conditions, was expressed as a fusion protein with ChSase ABC I. Results showed that the expression level and enzyme activity of GAPDH-ChSase ABC I were about 2.2 and 3.0 times higher than those of ChSase ABC I. By optimization of fermentation conditions, higher productivity of ChSase ABC I was achieved as 880 ± 61 IU/g wet cell weight compared with the reported ones. The optimal temperature and pH of GAPDH-ChSase ABC I were 40 °C and 7.5, respectively. GAPDH-ChSase ABC I had a kcat/Km of 131 ± 4.1 L/µmol s and the catalytic efficiency was decreased as compared to ChSase ABC I. The relative activity of GAPDH-ChSase ABC I remained 89% after being incubated at 30 °C for 180 min and the thermostability of ChSase ABC I was enhanced by GAPDH when it was incubated at 30, 35, 40 and 45 °C.

Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications.

Canine olfactory ensheathing cells from the olfactory mucosa can be engineered to produce active chondroitinase ABC.

A multitude of factors must be overcome following spinal cord injury (SCI) in order to achieve clinical improvement in patients. It is thought that by combining promising therapies these diverse factors could be combatted with the aim of producing an overall improvement in function. Chondroitin sulphate proteoglycans (CSPGs) present in the glial scar that forms following SCI present a significant block to axon regeneration. Digestion of CSPGs by chondroitinase ABC (ChABC) leads to axon regeneration, neuronal plasticity and functional improvement in preclinical models of SCI. However, the enzyme activity decays at body temperature within 24-72h, limiting the translational potential of ChABC as a therapy. Olfactory ensheathing cells (OECs) have shown huge promise as a cell transplant therapy in SCI. Their beneficial effects have been demonstrated in multiple small animal SCI models as well as in naturally occurring SCI in canine patients. In the present study, we have genetically modified canine OECs from the mucosa to constitutively produce enzymatically active ChABC. We have developed a lentiviral vector that can deliver a mammalian modified version of the ChABC gene to mammalian cells, including OECs. Enzyme production was quantified using the Morgan-Elson assay that detects the breakdown products of CSPG digestion in cell supernatants. We confirmed our findings by immunolabelling cell supernatant samples using Western blotting. OECs normal cell function was unaffected by genetic modification as demonstrated by normal microscopic morphology and the presence of the low affinity neurotrophin receptor (p75(NGF)) following viral transduction. We have developed the means to allow production of active ChABC in combination with a promising cell transplant therapy for SCI repair.