Enzymatic comparison of two homologous enzymes reveals N-terminal domain of chondroitinase ABC I regulates substrate selection and product generation.

Chondroitinase ABC-type I (CSase ABC I), which can digest both chondroitin sulfate (CS) and dermatan sulfate (DS) in an endolytic manner, is an essential tool in structural and functional studies of CS/DS. Although a few CSase ABC I have been identified from bacteria, the substrate-degrading pattern and regulatory mechanisms of them have rarely been investigated. Herein, two CSase ABC I, IM3796 and IM1634, were identified from the intestinal metagenome of CS-fed mice. They show high sequence homology (query coverage: 88.00%, percent identity: 90.10%) except for an extra peptide (Met1-His109) at the N-terminus in IM1634, but their enzymatic properties are very different. IM3796 prefers to degrade 6-O-sulfated GalNAc residue-enriched CS into tetra- and disaccharides. In contrast, IM1634 exhibits nearly a thousand times more activity than IM3796 and can completely digest CS/DS with various sulfation patterns to produce disaccharides, unlike most CSase ABC I. Structure modeling showed that IM3796 did not contain an N-terminal domain composed of two ß-sheets, which is found in IM1634 and other CSase ABC I. Furthermore, deletion of the N-terminal domain (Met1-His109) from IM1634 caused the enzymatic properties of the variant IM1634-T109 to be similar to those of IM3796, and conversely, grafting this domain to IM3796 increased the similarity of the variant IM3796-A109 to IM1634. In conclusion, the comparative study of the new CSase ABC I provides two unique tools for CS/DS-related studies and applications and, more importantly, reveals the critical role of the N-terminal domain in regulating the substrate binding and degradation of these enzymes.

Enzyme Kinetics Features of the Representative Engineered Recombinants of Chondroitinase ABC I.

Chondroitinase ABC I (cABC I) from Proteus vulgaris is an important enzyme in medicinal biotechnology due to its ability to help axon regeneration after spinal cord injury. Its practical application involves solving several problems at the molecular and cellular levels. Structurally, most residues at the C-terminal domain of cABC I are arranged as organized strands, and only a small fraction of residues have helical conformation. The structural and functional features of modified residues on two specific helix fragments have previously been reported. The single mutant M889K has been combined with L679S and L679D mutants to make enzyme variants containing simultaneously modified helix. Here, the pH stability and temperature-based analysis of the transition state structure for the catalysis reaction were investigated. We found that double mutant L679D/M889K is the better choice to use in physiological conditions due to its higher pH stability at physiological pH as well as its different optimum temperature as compared with the (wild-type) WT protein. According to Arrhenius's analysis, the values of the Gibbs free energy of the transition state (∆G#) are not changed upon mutation. However, the relative contribution and absolute values of the enthalpy and entropy change to the total value of ∆G#, varied between the WT and mutants.

Hungatella hathewayi, an Efficient Glycosaminoglycan-Degrading Firmicutes from Human Gut and Its Chondroitin ABC Exolyase with High Activity and Broad Substrate Specificity.

The degradation of glycosaminoglycans (GAGs) by intestinal bacteria is critical for their colonization in the human gut and the health of the host. Both colonic Bacteroides and Firmicutes have been reported to degrade GAGs; however, the enzymatic details of the latter remain largely unknown. Our bioinformatic analyses of fecal Firmicutes revealed that their genomes, especially Hungatella hathewayi strains, are an abundant source of putative GAG-specific catabolic enzymes. Subsequently, we isolated a Firmicutes strain, H. hathewayi N2-326, that can catabolize various GAGs. While H. hathewayi N2-326 was as efficient in utilizing chondroitin sulfate A (CSA) and dermatan sulfate as Bacteroides thetaiotaomicron, a well-characterized GAG degrader, it outperformed B. thetaiotaomicron in assimilating hyaluronic acid. Unlike B. thetaiotaomicron, H. hathewayi N2-326 could not utilize heparin. The chondroitin lyase activity of H. hathewayi N2-326 was found to be present predominantly in the culture supernatant. Genome sequence analysis revealed three putative GAG lyases, but only the HH-chondroitin ABC lyase was upregulated in the presence of CSA. In addition, five CAZyme gene clusters containing GAG metabolism genes were significantly upregulated when grown on CSA. Further characterization of the recombinant HH-chondroitin ABC lyase revealed that it cleaves GAGs predominantly in an exo-mode to produce unsaturated disaccharides as the primary hydrolytic product while exhibiting a higher specific activity than reported chondroitin ABC lyases. HH-chondroitin ABC lyase represents the first characterized chondroitin lyase from intestinal Firmicutes and offers a viable commercial option for the production of chondroitin, dermatan, and hyaluronan oligosaccharides and also for potential medical applications. IMPORTANCE An increased understanding of GAG metabolism by intestinal bacteria is critical in identifying the driving factors for the composition, modulation, and homeostasis of the human gut microbiota. In addition, GAG-depolymerizing polysaccharide lyases are highly desired enzymes for the production of GAG oligosaccharides and as therapeutics. At present, the dissection of the enzymatic machinery for GAG degradation is highly skewed toward Bacteroides. In this study, we have isolated an efficient GAG-degrading Firmicutes bacterium from human feces and characterized the first chondroitin ABC lyase from a Firmicutes, which complements the fundamental knowledge of GAG utilization in the human colon. The genomic and transcriptomic analysis of the bacterium shows that Firmicutes might use a distinct approach to catabolize GAGs from that used by Bacteroides. The high specific activity of the characterized chondroitin ABC lyase aids future attempts to develop a commercial chondroitinase for industrial and medicinal applications.

Comparing similar versions of a connecting helix on the structure of Chondroitinase ABC I.

Regarding the existence of similar helices on the structure of different proteins, recently, novel variants of Chondroitinase ABC I (cABC I) have been constructed, where a representative helix between two structural motifs in Chondroitinase ABC I from Proteus vulgaris has been replaced by similar versions of helices found in other proteins. The previous study has revealed that the structural features and the activity of double mutants M886A/G887E (inspired by the 30 S ribosomal protein S1 from Geminocystis herdmanii) and M889I/Q891K (inspired by the chondroitin lyase from Proteus mirabilis) is comparable with that of wild-type (WT) cABC I. Here, the kinetic parameters of the enzyme activity for the WT and double mutants were determined. Of the recombinant double mutants, M889I/Q891K gave the highest catalytic efficiency with the kcat/Km value of approximately 2.3-fold increase, as compared with the WT and M886A/G887E. Modeling of experimental data showed that the mechanism of the heat-induced structural alteration, and the enzyme-substrate complex formation, changed upon mutation. These natural versions of the connecting helix can be used as an efficient linker in protein engineering studies as well as those investigations involving the use of biological linkers.

Machine-Assisted Discovery of Chondroitinase ABC Complexes toward Sustained Neural Regeneration.

Among the many molecules that contribute to glial scarring, chondroitin sulfate proteoglycans (CSPGs) are known to be potent inhibitors of neuronal regeneration. Chondroitinase ABC (ChABC), a bacterial lyase, degrades the glycosaminoglycan (GAG) side chains of CSPGs and promotes tissue regeneration. However, ChABC is thermally unstable and loses all activity within a few hours at 37 °C under dilute conditions. To overcome this limitation, the discovery of a diverse set of tailor-made random copolymers that complex and stabilize ChABC at physiological temperature is reported. The copolymer designs, which are based on chain length and composition of the copolymers, are identified using an active machine learning paradigm, which involves iterative copolymer synthesis, testing for ChABC thermostability upon copolymer complexation, Gaussian process regression modeling, and Bayesian optimization. Copolymers are synthesized by automated PET-RAFT and thermostability of ChABC is assessed by retained enzyme activity (REA) after 24 h at 37 °C. Significant improvements in REA in three iterations of active learning are demonstrated while identifying exceptionally high-performing copolymers. Most remarkably, one designed copolymer promotes residual ChABC activity near 30%, even after one week and notably outperforms other common stabilization methods for ChABC. Together, these results highlight a promising pathway toward sustained tissue regeneration.

Substrate specificity of Chondroitinase ABC I based on analyses of biochemical reactions and crystal structures in complex with disaccharides.

Chondroitinase ABC I (cABC-I) is the enzyme which cleaves the ß-1,4 glycosidic linkage of chondroitin sulfate (CS) by ß-elimination. To elucidate more accurately the substrate specificity of cABC-I, we evaluated the kinetic parameters of cABC-I and its reactivity with CS isomers displaying less structural heterogeneity as substrates, e.g., approximately 90 percent of disaccharide units in Chondroitin sulfate A (CSA) or Chondroitin sulfate C (CSC) is D-glucuronic acid and 4-O-sulfated N-acetyl galactosamine (GalNAc) (A-unit) or D-glucuronic acid and 6-O-sulfated GalNAc (C-unit), respectively. cABC-I showed the highest reactivity to CSA and CSC among all CS isomers, and the kcat/Km of cABC-I was higher for CSA than for CSC. Next, we determined the crystal structures of cABC-I in complex with CS disaccharides, and analyzed the crystallographic data in combination with molecular docking data. Arg500 interacts with 4-O-sulfated and 6-O-sulfated GalNAc residues. The distance between Arg500 and the 4-O-sulfate group was 0.8 Å shorter than that between Arg500 and the 6-O-sulfated group. Moreover, it is likely that the 6-O-sulfated group is electrostatically repulsed by the nearby Asp490. Thus, we demonstrated that cABC-I has the highest affinity for the CSA richest in 4-O-sulfated GalNAc residues among all CS isomers. Recently, cABC-I was used to treat lumbar disc herniation. The results provide useful information to understand the mechanism of the pharmacological action of cABC-I.

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.

Preparation of low-molecular-weight chondroitin sulfates by complex enzyme hydrolysis and their antioxidant activities.

Chondroitin sulfate (CS) has attracted widespread attention because of its numerous pharmacological activities. Low-molecular-weight chondroitin sulfates (LMWCSs) derived from the degradation of CS are reported to have better biological properties than whole CS. In this study, to obtain LMWCSs with high antioxidant activity, we depolymerized CS using complex enzymes, namely, chondroitinase ABC I (ChSase ABC I) and ChSase ABC II. The conditions of the complex enzyme hydrolysis (CEH) were optimized, and the structures and antioxidant activities of CS and LMWCSs were investigated. The results showed that the CEH conditions enhanced the antioxidant activities of the products as compared to CS. The basic structures of the LMWCSs and sulfate groups were well preserved after hydrolysis. Therefore, CEH provides an efficient and safe strategy to obtain LMWCSs, which can be used in antioxidant drugs, healthy foods, and cosmetics.

A combinatorial approach for spinal cord injury repair using multifunctional collagen-based matrices: development, characterization and impact on cell adhesion and axonal growth.

Spinal cord injury is a devastating condition of the central nervous system, in which traditional treatments are largely ineffective due to the complex nature of the injured tissue. Therefore, biomaterial-based systems have been developed as possible alternative strategies to repair the damaged tissue. In the present study, we aimed to design bioactive agent loaded scaffolds composed of two layers with distinct physical properties to improve tissue regeneration. An electrospun layer with aligned nanofibers was made of collagen (Col) Type-I, poly(lactide-co-glycolide) (PLGA) and laminin to promote cell attachment of mesenchymal-like stem cells towards the direction of fibers, while a Col-based second layer was fabricated by plastic compression to act as a releasing system for NT-3 and chondroitinase ABC, so that axonal growth could be stimulated. Results showed that a source of mesenchymal stem cell (MSC)-like cells, adipose tissue-derived stem cells cultured on the fibrous layer of the matrices were able to adhere and proliferate, where the aligned fibers promoted the cell growth in an organized way. Furthermore, the bilayered matrices also promoted dorsal root ganglion neurite outgrowth. The bilayered matrice with Col/PLGA + laminin top layer appears to promote higher neurite growth. Collectively, the designed constructs show promising structural properties and biological performance for being employed as a scaffold for engineering the spinal cord tissue.

Enhancing myelin repair in experimental model of multiple sclerosis using immobilized chondroitinase ABC I on porous silicon nanoparticles.

Removal of chondroitin sulfate proteoglycans (CSPGs) with chondroitinase ABC I (ChABC) facilitates axonal plasticity, axonal regeneration and remyelination, following injury to the central nervous system (CNS). However, the ChABC rapidly undergoes thermal inactivity and needs to be injected repeatedly. Here this limitation was overcame by immobilizing the ChABC on porous silicon (PSi) nanoparticles (ChABC@PSi). The efficacy of immobilized ChABC on CSPGs level and the demyelination insult was assessed in mice corpora callosa demyelinated by 6 weeks cuprizone (CPZ) feeding. ChABC@PSi was able to reduce the amount of CSPGs two weeks after animals treatment. CSPGs digestion by ChABC@PSi reduced the extent of demyelinated area as well as the astrogliosis. Furthermore, ChABC@PSi treatment increased the number of newly generated oligodendrocyte lineage cells which imply for enhanced myelin repair. Our results showed that effective CSPGs digestion by ChABC@PSi enhanced remyelination in CPZ model. Accordingly, ChABC@PSi may have a great potential to be used for treatment of diseases like multiple sclerosis and spinal cord injury by promoting the regeneration of damaged nerves.

Examination of chondroitinase ABC I immobilization onto dextran-coated Fe3O4 nanoparticles and its in-vitro release.

Chondroitinase ABC I (cABC I) has received notable attention in treatment of spinal cord injuries and its application as therapeutics has been limited due to low thermal stability at physiological temperature. In this study, cABC I enzyme was immobilized on the dextran-coated Fe3O4 nanoparticles through physical adsorption to improve the thermal stability. The nanoparticles were characterized using XRD, SEM, VSM, and FTIR analyses. Response surface methodology and central composite design were employed to assess factors affecting the activity of immobilized cABC I. Experimental results showed that pH 6.3, temperature 24 °C, enzyme/support mass ratio 1.27, and incubation time 5.7 h were the optimal immobilization conditions. It was found that thermal stability of immobilized cABC I was significantly improved. In-vitro cABC I release was studied under pH 7.5 and temperature 37 °C and the results indicated that 70 % release occurred after 9 h and the release mechanism was first-order kinetic model.

ChABC-loaded PLGA nanoparticles: A comprehensive study on biocompatibility, functional recovery, and axonal regeneration in animal model of spinal cord injury.

Spinal Cord Injury (SCI) is one of the leading causes of physical disability. In this study, spherical PLGA nanoparticles (NPs) containing ChABC enzyme were manufactured and fully characterized for SCI therapy. The NPs were used in the rat's contused spinal cord to assess the functional improvement and scar digestion. Twenty-three adult male Wistar rats (275 ± 25 g) were assigned into four groups of control, sham, blank-treated particle, and ChABC-treated particle. Throughout the survey, the BBB scores were obtained for all the groups. Finally, the injured sections of animals were dissected, and histological studies were conducted using Luxol fast blue and Bielschowsky. The biocompatibility and non-toxicity effects of the NPs on olfactory ensheathing cells (OECs) were confirmed by the MTT test. The flow-cytometry revealed the purity of cultured OECs with p75+/GFAP+ at around 87.9 ± 2.4%. Animals in the control and the blank-treated groups exhibited significantly lower BBB scores compared with the ChABC-treated particle group. Histological results confirmed the induced contusion models in the injured site. Myelin was observed in the treated groups, especially when the ChABC-loaded nanoparticles were utilized. The immunohistochemistry results indicated the scar glial degradation in animals treated by the ChABC-loaded particles. According to this study, the loaded particles can potentially serve as a suitable candidate for spinal cord repair, functional recovery and axonal regeneration.

Hyaluronic acid of tailored molecular weight by enzymatic and acid depolymerization.

Hyaluronic acid (HA) is a glycosaminoglycan crucial for the homeostasis of tissues, and its role on cell signalling and regulation of tissue injury and repair largely depends on HA molecular weight. Therefore, HA application in a variety of fields requires HA of defined size. While a number of enzymatic, chemical and physical methods exist for HA depolymerization, limited information is currently available for accurate planning of experiments. In the present work, we propose a pseudo-mechanistic model to describe depolymerization kinetics of HA with hyaluronidase, chondroitinase ABC and phosphoric acid. Data to feed the model was provided by monitoring molecular weight reduction by gel permeation chromatography with light scattering detection over 24 h. Five enzyme to substrate ratios and three temperatures were used for enzymatic and chemical reactions respectively, allowing for selection of operational parameters in a range of conditions. The model adequately reproduces the resulting data providing flexibility in the planning of the reactions to obtain HA of the desired molecular weight.

Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate.

Chondroitinase ABC I (ChSase ABC I) is a key enzyme of chondroitin sulfate (CS) degradation and widely used for CS detection in the medicine filed. However, the recombinant ChSase ABC I was weakly expressed in Escherichia coli because the forms of it were mostly inclusion bodies. In this study, a signal peptide (pelB) was used for the soluble form expression of ChSase ABC I in E. coli. Then the culture condition for ChSase ABC I expression was optimized through response surface methodology. Results revealed that the expression level of ChSase ABC I in a 7.5 L fermentor (29.03 mL-1) was approximately 1.65-fold higher than that of the shake flask level (17.55 mL-1). The enzymatic properties and kinetic constants of recombinant ChSase ABC I were also studied. Recombinant ChSase ABC I was also used to detect the specific disaccharides content of CS from different sources. This study not only eliminates the problem of the enzyme expressed as an inclusion body, but also solves the current problem of expensive ChSase ABC. In a word, it would be an ideal strategy for ChSase ABC high-efficiency expression and a great method to detect specific disaccharides of CS in biomedical field.

Neurogenic Niche Conversion Strategy Induces Migration and Functional Neuronal Differentiation of Neural Precursor Cells Following Brain Injury.

Glial scars formed after brain injuries provide permissive cues for endogenous neural precursor/stem cells (eNP/SCs) to undergo astrogenesis rather than neurogenesis. Following brain injury, eNP/SCs from the subventricular zone leave their niche, migrate to the injured cortex, and differentiate into reactive astrocytes that contribute to glial scar formation. In vivo neuronal reprogramming, directly converting non-neuronal cells such as reactive astrocytes or NG2 glia into neurons, has greatly improved brain injury repair strategies. However, reprogramming carries a high risk of future clinical applications such as tumorigenicity, involving virus. In this study, we constructed a neural matrix to alter the adverse niche at the injured cortex, enabling eNP/SCs to differentiate into functional neurons. We found that the neural matrix functioned as a "glial trap" that largely concentrated and limited reactive astrocytes to the core of the lesion area, thus altering the adverse niche. The eNP/SCs migrated toward the injured cortex and differentiated into functional neurons. In addition, regenerated neurites extended across the boundary of the injured cortex. Mice treated with the neural matrix demonstrated significant behavioral recovery. For the first time, we induced eNP/SC-derived functional neurons in the cortex after brain injury without the use of viruses, microRNAs, or small molecules. Our novel strategy of applying this "glial trap" to obtain functional neurons in the injured cortex may provide a safer and more natural therapeutic alternative to reprogramming in future clinical applications.

Marine chondroitin sulfate of defined molecular weight by enzymatic depolymerization.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan with diverse biological activities, which are influenced by molecular weight (Mw) and sulfation pattern. In the present work, we take advantage of the characteristic high Mw of fish CS (51-70 kDa) to obtain lower Mw fragments with hyaluronidase and chondroitinase ABC. With this aim, we present a pseudo-mechanistic model capable of reproducing the decrease in Mw of CS from five different fish species over 24 h at four enzyme to substrate ratios. The fitting parameters of the model for each species allow to establish conditions of reaction to produce CS of the desired Mw. Furthermore, the main features of the sulfation pattern of fish CS remain in the depolymerized fragments, highlighting the feasibility of the proposed approach.

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.

Proteoglycan removal by chondroitinase ABC improves injectable collagen gel adhesion to annulus fibrosus.

Intervertebral disc (IVD) herniations are currently treated with interventions that leave the IVD with persistent lesions prone to further herniations. Annulus fibrosus (AF) repair has become of interest as a method to seal defects in the IVD and prevent reherniation, but this requires strong adhesion of the implanted biomaterial to the native AF tissue. Our group has previously developed a high-density collagen (HDC) gel for AF repair and tested its efficacy in vivo, but its adhesion to the AF could be improved. Increased cell adhesion to cartilage has previously been reported through chondroitinase ABC (ChABC) digestion, which removes proteoglycans and increases access to cell binding motifs. Such approaches could also increase biomaterial adhesion to tissue, but the effects of ChABC digestion on AF have yet to be investigated. In this study, ovine AF tissue was digested with either 10 U/mL ChABC or saline for up to 10 min and the effect of this treatment on collagen adhesion between AF tissue samples was investigated by histology and mechanical testing in a lap-shear configuration. ChABC digestion removed proteoglycans within the AF in a time-dependent fashion and enhanced adhesion of the HDC gel to the AF. ChABC digestion increased the elastic toughness and total shear energy of the HDC gel-AF interface by 88% and 46% respectively. ChABC treatment enhanced the adhesion of the HDC gel to the AF without significantly decreasing native AF cell viability. Thus, ChABC digestion is a viable method to improve adhesion of biomaterials for AF repair. STATEMENT OF SIGNIFICANCE: Intervertebral disc herniations are currently treated with interventions that leave persistent lesions in the annulus fibrosus that are prone to further herniations. Annular repair is a promising method to seal lesions and prevent reherniation, but requires strong adhesion of the implanted biomaterial to native annulus fibrosus. Since large proteoglycans like aggrecan occupy regions of the extracellular matrix between collagen fibers in the annulus fibrosus, we hypothesized that removing proteoglycans via chondroitinase digestion would increase the adhesion of annular repair hydrogels. This investigation demonstrated that chondroitinase removed proteoglycans within annulus fibrosus tissue, enhanced the interaction of an injected collagen gel with the native tissue, and mechanically improved adhesion between the collagen gel and annulus fibrosus. This is the first study of its kind to evaluate the biochemical and mechanical effects of short-term chondroitinase digestion on annulus fibrosus tissue.

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.