Severian Dumitriu

Inclusion and release of proteins from polysaccharide-based polyion complexes

The notion of a polyelectrolyte complex is well established for the complexation of two polymers one anionic, the other cationic. Electronic microscopy studies have shown the formation of a fibrilar structure. A method for the preparation of polyionic hydrogels from the complexation of chitosan and xanthan is reported. Electronic microscopy studies have shown the formation of a fibrilar structure. Stable hydrogels have been used to immobilize xylanase, lipase and protease. The immobilized xylanase and lipase activity was significantly higher than that of the free enzyme.

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Study of biodegradation behavior of chitosan-xanthan microspheres in simulated physiological media.

Microspheres of a polyelectrolyte complex hydrogel were prepared from chitosan and xanthan after interaction between the two polyionic polymers. Their biodegradation was studied vs. chitosan. Simulated gastric fluid (SGF, pH 1.2) and intestinal fluid (SIF, pH 7.5) both as biodegradation media and phosphate buffered saline (PBS, pH 7.4) as a negative control were used. The degradation studies were performed at 37 degrees C at 240 rpm permanent stirring to mimic the physiologic conditions. High performance liquid chromatography (HPLC) was carried out to quantify the chitosan degradation products using glucosamine (GA) and N-acetyl-D-glucosamine (N-Ac-GA) as references. The peaks area integration method was used to determine the amount of each degradation product as a function of incubation time in the media. The effect of the media on the morphological structure of microspheres was assessed by scanning electron microscopy. From HPLC studies, it appeared that in SGF and SIF the major degradation products were glucosamine (GA) and N-acetyl-D-glucosamine (NAc-GA). In the first 15 days, oligochitosan fractions were released from the complex, whereas N-acetyl-D-glucosamine was detected in the media after this period. The degradation kinetics were assessed by the measurement of the cumulative degradation products, which showed faster degradation of chitosan than the complex in SGF and SIF. SEM micrographs showed an enhancement of microsphere porosity as a function of incubation time in the simulated physiological media. Our results suggest a better control of the degradation kinetics when chitosan is complexed to xanthan.

Immobilization of Xylanase in Chitosan−Xanthan Hydrogels

endo‐1,4‐β‐Xylanase (E.C. 3.2.1.8) has been immobilized in hydrogel beads prepared by complexation between chitosan and xanthan. The enzyme immobilization efficiency is between 88 and 98% within a wide range of concentrations of xylanase. The optimum xylanase activity requires a pH between 5.0 and 5.5. The immobilized enzymes show 60–70% higher activity than free enzymes. The Km of the immobilized xylanase increases with the concentration of the enzyme in the beads. The latter show, by electron microscopy, a predominantly fibrilar microstructure within which there are regions having globular shapes where the enzymes are lodged.

Polyionic Hydrogels Obtained by Complexation between Xanthan and Chitosan: Their Properties as Supports for Enzyme Immobilization

A method for the preparation of hydrogels from the complexation of chitosan and xanthan is reported. Stable hydrogels capable of retaining be tween 65 and 95% weight water were prepared. The water retention and prop erties of the hydrogels were studied as a function of the degree of acetylation of chitosan and the ratio chitosan/xanthan used in the preparation of the gel. Spectroscopic FTIR was used to confirm complexation between the amine (chi tosan) and carboxylic (xanthan) groups. Electron micrographs (SEM and TEM) show the formation of a fibrillar structure with characteristic pore sizes be tween 100 and 1000 nm and fibril diameters between 50 and 100 nm. The diffu sion coefficient of 4-O-methyl-d-glucurono-D-xylan Remazol Brilliant Blue R (RBB-xylan) in the complex chitosan-xanthan was 2.02 × 10-12 m 2s-1 at 30°C. The chitosan-xanthan complex was used to immobilize two enzymes (endo-1,4- β-xylanase and protease) either as single enzymes or as a binary system. Immo bilization varied between 85 and 98%. The immobilized xylanase activity was significantly greater with respect to the free enzyme while the binary enzyme system promoted protease activity.

Lipase immobilization into porous chitoxan beads : Activities in aqueous and organic media and lipase localization

Lipases were noncovalently immobilized in Chitoxan, a polyionic hydrogel obtained by complexation between chitosan and xanthan. The properties of free and immobilized lipases have been compared. In the aqueous medium, the activity was twice as high for immobilized lipases as for free lipases. Immobilized lipases in chitoxan were able to hydrolyze triacylglycerols in three distinct organic solvent media. At the microstructural level, lipases were not distributed uniformly in the chitoxan beads. Higher concentrations of lipase were found in the outer membrane‐like layer of the beads, as compared with lower concentrations in the inner part of the beads.

Immobilization of Enzymes into a Polyionic Hydrogel: ChitoXan

Three enzymes were immobilized onto polyionic hydrogel, ChitoXan, obtained by complexation between chitosan and xanthan. The biocatalysts used were two proteases (protease type XIX from Fungal d’Aspergillus sojae and the trypsin type II.S from Porcine Pancreas) and a lipase (lipase Type VII from Candida rugosa). The immobilization efficiencies and the relative activities were investigated for these enzymes. The immobilization efficiencies changed with each enzyme and varied between 53 and 80%. Good relative activities were found for the lipase Type VII from Candida rugosa and the protease type XIX from Fungal d’Aspergillus sojae. For the latter, the influence of several factors were studied: molarity of the storage buffer, storage temperature and time of hydrogel, and the enzyme concentration. For the immobilized lipase, hydrolysis of olive oil in aqueous and organic media has been compared. This study confirmed that the lipase modified the external and internal structure of the hydrogel from fibrillar to the formation of globular structures in the presence of lipases.

Use of a drug eluting pleural catheter for pleurodesis

ABSTRACT Purpose: Repeated administration of low-dose silver nitrate (SN) has been shown to be effective in creating pleurodesis. This study aimed to determine the effectiveness of a SN-eluting pleural catheter for pleurodesis. Methods: Catheters with a chitosan—SN—hyaluronic acid hydrogel coating designed to release SN over 14 days, or placebo uncoated catheters, were inserted in rabbit and lamb pleurodesis models. Pleurodesis was assessed at 28 days according to a 1–8 point scoring system and pleural fibrosis and inflammation assessed histologically on a 0–4 point scale. Results: In the rabbit model, pleurodesis scores were significantly increased in both the 24 mg and 50 mg SN catheters versus control animals as well as compared to the contralateral untreated pleural space (median-treated side scores were 5, 8, and 1, respectively, median score for contralateral side was 1 in all groups). In the lamb model, pleurodesis scores were significantly increased in both the 750 mg and 1000 mg catheter groups versus control animals as well as compared to the contralateral untreated pleural space (median-treated side scores were 7, 7, and 1, respectively, median score for contralateral pleural space was 1 in all groups). Catheters appeared well tolerated, although higher than expected mortality was seen in the 50 mg catheter rabbit group. Conclusions: A catheter designed to deliver SN to the pleural space over 14 days appears to be effective in creating pleurodesis. Further investigations to determine in-vivo catheter pharmacokinetics, toxicity, dose and optimal coating methods are warranted.

Polyionic hydrogels as support for immobilization of lipase

Polyionic hydrogels have been prepared by complexation of chitosan and xanthan. These hydrogels have been used to immobilize lipase from porcine pancreas (E.C. 3.1.1.3). Immobilization efficiency varied between 90 and 99% of initial activity. Immobilized lipase retained its activity towards hydrolysis of olive oil in water as an emulsion and of olive oil in isooctane.

Processes with immobilized enzymes and cells

The immobilization of enzymes is a technique extensively studied since the late 1960s (Silman and Katchalski, 1966). The knowledge base accumulated on enzyme and cells immobilization studies has grown to very large proportions (Klibanov, 1983; Ariga et al., 1993; Crumbliss et al., 1993; Champagne et al., 1994). This wealth of information is one of the primary reasons for the present advances in enzyme engineering. The introduction of immobilized enzyme systems into commercial use, which was slower than predicted, has been the result of numerous factors, such as the long time required for approval of new processes for use in food applications, the need to control microbial contamination in biological reactor systems and some enzyme characteristics that limit the economic success of the immobilization process. The engineering of enzymes with better characteristics will overcome some of the problems encountered that have prevented commercial processes from developing.