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.

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.

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.

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.

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.

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 the 5hmC specific endonuclease PvuRts1I.

PvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 Å resolution. Although the protein has been crystallized in the absence of DNA, the structure is very informative. It shows that PvuRts1I consists of an N-terminal, atypical PD-(D/E)XK catalytic domain and a C-terminal SRA domain that might accommodate a flipped 5hmC or 5ghmC base. Changes to predicted catalytic residues of the PD-(D/E)XK domain or to the putative pocket for a flipped base abolish catalytic activity. Surprisingly, fluorescence changes indicative of base flipping are not observed when PvuRts1I is added to DNA substrates containing pyrrolocytosine in place of 5hmC (5ghmC). Despite this caveat, the structure suggests a model for PvuRts1I activity and presents opportunities for protein engineering to alter the enzyme properties for biotechnological applications.

A structural view of the dissociation of Escherichia coli tryptophanase.

Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.

Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: an insight from Ramachandran plot.

The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein.

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.

Substrate promiscuity in DNA methyltransferase M.PvuII. A mechanistic insight.

M.PvuII is a DNA methyltransferase from the bacterium Proteus vulgaris that catalyzes methylation of cytosine at the N4 position. This enzyme also displays promiscuous activity catalyzing methylation of adenine at the N6 position. In this work we use QM/MM methods to investigate the reaction mechanism of this promiscuous activity. We found that N6 methylation in M.PvuII takes place by means of a stepwise mechanism in which deprotonation of the exocyclic amino group is followed by the methyl transfer. Deprotonation involves two residues of the active site, Ser53 and Asp96, while methylation takes place directly from the AdoMet cofactor to the target nitrogen atom. The same reaction mechanism was described for cytosine methylation in the same enzyme, while the reversal timing, that is methylation followed by deprotonation, has been described in M.TaqI, an enzyme that catalyzes the N6-adenine DNA methylation from Thermus aquaticus. These mechanistic findings can be useful to understand the evolutionary paths followed by N-methyltransferases.

Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase.

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator.

BACKGROUND: Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. RESULTS: Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans). In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting the putative hairpins had no significant effects. CONCLUSIONS: The initiation of translation of pvuIIR appears to be independent of that for pvuIIC. Direct tests failed to detect regulatory rules for either gene overlap or the putative hairpins. Thus, at least during balanced growth, transcriptional control appears to be sufficiently robust for proper regulation of this RM system.

Overexpression and characterization in Bacillus subtilis of a positionally nonspecific lipase from Proteus vulgaris.

A Proteus vulgaris strain named T6 which produced lipase (PVL) with nonpositional specificity had been isolated in our laboratory. To produce the lipase in large quantities, we cloned its gene, which had an opening reading frame of 864 base pairs and encoded a deduced 287-amino-acid protein. The PVL gene was inserted into the Escherichia coli expression vector pET-DsbA, and active lipase was expressed in E. coli BL21 cells. The secretive expression of PVL gene in Bacillus subtilis was examined. Three vectors, i.e., pMM1525 (xylose-inducible), pMMP43 (constitutive vector, derivative of pMM1525), and pHPQ (sucrose-inducible, constructed based on pHB201), were used to produce lipase in B. subtilis. Recombinant B. subtilis WB800 cells harboring the pHPQ-PVL plasmid could synthesize and secrete the PVL protein in high yield. The lipase activity reached 356.8 U/mL after induction with sucrose for 72 h in shake-flask culture, representing a 12-fold increase over the native lipase activity in P. vulgaris. The characteristics of the heterologously expressed lipase were identical to those of the native one.

[Cloning and expression of non-position-specific lipase gene from Proteus vulgaris].

OBJECTIVE: To produce Proteus vulgaris lipase (PVL) in large quantities, we cloned and expressed the lipase gene in Escherichia coli. METHODS: We cloned PVL gene by PCR method and then inserted the open reading frame of PVL gene into pET-DsbA and pMBP-P vectors. PVL gene was expressed in E. coli with the introduction of isopropyl beta-D-1-thiogalactopyranoside (IPTG). We also studied the optimal culture conditions, including the concentrations of glucose, IPTG and ampicillin, induction temperature, and pH value of the medium. The characteristics of recombinant lipase were examined after affinitive purification by His-chelating affinity chromatography. RESULTS: The open reading frame of PVL gene consisted of 864 base pairs, encoding a protein of 287 amino acids. The sequence was deposited to GenBank with the accession number FJ643627. The gene was expressed in E. coli and active lipase was obtained from E. coli BL21 cells by the induction of IPTG, and the lipase production reached 192. 2 U/mL in BL21 [pET-PVL] after culture for 15 h at 15 degrees C. The maximum production was obtained by culturing BL21 cells in LB medium (pH 8.5) with 15 mg/mL glucose and 200 mg/L ampicillin, as well as adding 100 mg/L IPTG at OD600 of 1.2. A single protein band of 31 kDa was displayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after affinitive purification. The properties of lipase expressed in E. coli were similar to the native one, which could hydrolyze all three esters of triglyceride. CONCLUSION: We have succeeded in over-expressing the lipase gene from P. vulgaris in E. coli, and this research has laid a foundation for improvement and industrial application of this lipase.

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.

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.

Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern.

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.

Regulatory circuit based on autogenous activation-repression: roles of C-boxes and spacer sequences in control of the PvuII restriction-modification system.

Type II restriction-modification (R-M) systems comprise a restriction endonuclease (REase) and a protective methyltransferase (MTase). After R-M genes enter a new cell, MTase must appear before REase or the chromosome will be cleaved. PvuII and some other R-M systems achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator (the controlling or 'C' protein C.PvuII). This study reveals, through in vivo titration, that C.PvuII is not only an activator but also a repressor for its own gene. In other systems, this type of circuit can result in oscillatory behavior. Despite the use of identical, symmetrical C protein-binding sequences (C-boxes) in the left and right operators, C.PvuII showed higher in vitro affinity for O(L) than for O(R), implicating the spacer sequences in this difference. Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation. A nonrepressing mutant exhibited poor establishment in new cells. Comparing promoter-operator regions from PvuII and 29 R-M systems controlled by C proteins revealed that the most-highly conserved sequence is the tetranucleotide spacer separating O(L) from O(R). Any changes in that spacer reduced the stability of C.PvuII-operator complexes and abolished activation.

In vitro evaluation of chondroitin sulphate-chitosan microspheres as carrier for the delivery of proteins.

A novel formulation based on chondroitin sulphate/chitosan microspheres (CS/CH) has been investigated for oral delivery of macromolecules using ovalbumin as the model protein (OVA). The microspheres were prepared by a new emulsion-complex coacervation method. Physico-chemical properties of the polymers constituting microparticulate matrix were investigated by IR, DSC, TGA and X-ray diffraction analyses. In vitro tests were performed to evaluate the drug delivery system degradation and the protein release under conditions simulating the intestinal fluids. The ability of colonic enzymes to degrade the microparticulate systems was simulated employing the chondroitinase ABC enzyme. Results showed that the different CS/CH compositions influenced both microparticles stability and the protein release rate. Only the microspheres composed by 1:1 chondroitin sulphate-chitosan ratio achieved an OVA release profile suitable to a possible colon targeting. These microspheres released approximately 30% of ovalbumin encapsulated in 24 h in the different aqueous media tested, while they released 100% of protein in the presence of chondroitinase. The preliminary results demonstrated that chondroitin sulphate-chitosan microspheres can be a suitable delivery system for protein drug envisaged to oral administration.