Effect of immobilized neutral protease on the preparation and physicochemical properties of low molecular weight chitosan and chito‐oligomers

Neutral protease was immobilized on glutaraldehyde‐pretreated N‐succinyl chitosan hydrogel beads and the biocatalyst obtained was used for the preparation of low molecular weight chitosan and chito‐oligomers with molecular weight of 1.9–23.5 kDa from commercial chitosan. Factors affecting the chitinolytic hydrolysis were described. The degradation was monitored by gel permeation chromatography. The structure of degraded chitosan was characterized by Fourier transform infrared, X‐ray diffraction and liquid chromatography‐mass spectrometry. Immobilized neutral protease showed optimal depolymerization at pH 5.7 and 50°C. The degree of deacetylation of the hydrolysates did not change compared to that of the initial chitosan. The decrease of molecular weight led to transformation of crystal structure but the chemical structures of residues were not modified. The degree of polymerization of chito‐oligomers was mainly from 3 to 8. The method allows cyclic procedures of immobilized enzyme and N‐succinyl chitosan support utilization, and is suitable for a large‐scale production of the low molecular weight chitosan and chito‐oligomers free of protein admixtures. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102:4185–4193, 2006     

Calcium‐carboxymethyl chitosan hydrogel beads for protein drug delivery system

In this study, carboxymethyl chitosan (CMC) hydrogel beads were prepared by crosslinking with Ca²⁺. The pH‐sensitive characteristics of the beads were investigated by simulating gastrointestinal pH conditions. As a potential protein drug delivery system, the beads were loaded with a model protein (bovine serum albumin, BSA). To improve the entrapment efficiency of BSA, the beads were further coated with a chitosan/CMC polyelectrolyte complex (PEC) membrane by extruding a CMC/BSA solution into a CaCl₂/chitosan gelation medium. Finally, the release studies of BSA‐loaded beads were conducted. We found that, the maximum swelling ratios of the beads at pH 7.4 (17–21) were much higher than those at pH 1.2 (2–2.5). Higher entrapment efficiency (73.2%) was achieved in the chitosan‐coated calcium‐CMC beads, compared with that (44.4%) in the bare calcium‐CMC beads. The PEC membrane limited the BSA release, while the final disintegration of beads at pH 7.4 still leaded to a full BSA release. Therefore, the chitosan‐coated calcium‐CMC hydrogel beads with higher entrapment efficiency and proper protein release properties were a promising protein drug carrier for the site‐specific release in the intestine. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3164–3168, 2007     

低分子量壳聚糖的制备

利用H2O2的强氧化性制备低分子量分布的壳聚糖是将虾皮用HC l浸泡去除碳酸钙盐;再用稀碱除去蛋白质得甲壳素;然后浓碱在50℃与其反应,并控制反应时间,分别制备出脱乙酰度为85%,93%,99%的壳聚糖,最后用H2O2氧化不同脱乙酰化壳聚糖,得到不同低分子量的壳聚糖。其中,脱乙酰度为85%壳聚糖用不同浓度的H2O2降解,得到了4.7×105,3.5×105,2.5×105,1.2×105,8×105等5个不同分子量段的壳聚糖产品。H2O2浓度越大,降解所得壳聚糖的分子量就越小。     

Synthesis of geranyl butyrate with the poly(acrylic acid‐co‐hydroxy propyl methacrylate‐cl‐ethylene glycol dimethacrylate) hydrogel immobilized lipase of Pseudomonas aeruginosa MTCC‐4713

Microbial lipases (E.C. 3.1.1.3) are the preferred biocatalysts for the synthesis of various fragrance compounds, such as linalool acetate, citronellal acetate, and geranyl acetate, in organic solvents over chemical synthesis. In this study, a purified alkaline extracellular lipase of Pseudomonas aeruginosa MTCC‐4713 was efficiently immobilized onto a synthetic poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel by surface adsorption, and the bound lipase was evaluated for its hydrolytic potential toward various p‐nitrophenyl acyl esters, which differed in their C‐chain length. Among four series of hydrogels prepared by the variation of the concentrations of monomer and crosslinker, two hydrogels, namely, I_5d and I_20d, that exhibited relatively higher protein (lipase activity) bindings were selected to perform hydrolytic and synthetic (geranyl butyrate) reactions in aqueous and organic solvents. The hydrogel‐bound lipase was highly hydrolytic toward p‐nitrophenyl ester (C: 16; p‐nitrophenyl palmitate). The hydrogel‐immobilized lipase was quite stable and retained approximately 57.6% of its original hydrolytic activity after the fifth cycle of reuse under optimized conditions (pH 8.5, 65°C). The hydrogel‐immobilized lipase when used to perform the esterification of geraniol/butyric acid (400 : 100 mM) in n‐heptane resulted in 98.8 mM geranyl butyrate at 65°C under shaking (120 rpm) after 15 h of reaction time. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 100 mg per reaction volume (1 mL) resulted in the complete conversion of the reactants into geranyl butyrate. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

低聚壳聚糖的复合酶法制备研究

通过对黏均分子量、酶活力、水解率和还原糖浓度的测定,研究了由商业α-淀粉酶、纤维素酶和果胶酶1∶1∶1(m/m/m)组成的复合酶降解壳聚糖的最佳工艺条件,结果表明:复合酶在酶底物比为1∶5(m/m)、pH 5.3、温度56℃的条件下,酶解2 h可得到分子量为1 000～4 000的低聚壳聚糖,且通过傅里叶红外光谱分析酶解后的产物结构无明显变化。     

Preparation and in vitro evaluation of new pH‐sensitive hydrogel beads for oral delivery of protein drugs

New biodegradable pH‐responsive hydrogel beads based on chemically modified chitosan and sodium alginate were prepared and characterized for the controlled release study of protein drugs in the small intestine. The ionotropic gelation reaction was carried out under mild aqueous conditions, which should be appropriate for the retention of the biological activity of an uploaded protein drug. The equilibrium swelling studies were carried out for the hydrogel beads at 37°C in simulated gastric (SGF) and simulated intestinal (SIF) fluids. Bovine serum albumin (BSA), a model for protein drugs was entrapped in the hydrogels and the in vitro drug release profiles were established at 37°C in SGF and SIF. The preliminary investigation of the hydrogel beads prepared in this study showed high entrapment efficiency (up to 97%) and promising release profiles of BSA. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Immobilization of lipase with alginate hydrogel beads and the lipase‐catalyzed kinetic resolution of α‐phenyl ethanol

The immobilization of enzymes is one of the key issues in both the field of enzymatic research and industrialization. In this article, we report a facile method for immobilizing Candida antarctica lipase B in an alginate carrier. In the presence of calcium cations, an enzyme–alginate suspension was crosslinked to form beads with a porous structure at room temperature, and the enzymes were well dispersed in the beads. The chiral resolution of α‐phenyl ethanol in the organic phase was tested by the enzyme–alginate beads. The effects of the reaction parameters, such as the enzyme concentration, temperature, and molar ratio of the substrate to the solvent, on the resolution behavior are discussed. Reuse cycle experiments for the chiral resolution of α‐phenyl ethanol demonstrated that the activity of the enzyme–alginate beads was maintained without marked deactivation up to five repeated cycles. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40178.     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

Preparation and rheology of titanium dioxide nanoparticles loaded κ-carrageenan hydrogel beads strengthened by mixed salts for multipollutant water remediation

Hydrogel beads are emerging as alternate adsorbent material for the batch or continuous column treatment of dye wastewater. Polysaccharide-based gel beads are preferred for their uniform shape and size, large specific surface area, and easy separation by filtration and subsequent reuse. In this work, we prepare and rheologically characterize UV-active κ-carrageenan/TiO₂ beads for the adsorption and degradation of dyes. The 3–4 mm sized nanocomposite beads are formed by the extrusion of κ-carrageenan/TiO₂ gel solution in salt solutions. The maximum increase in modulus was observed with the addition of 1% w/v TiO₂ to κ-carrageenan and subsequent crosslinking with mixed salt of KCl and CaCl₂. κ-carrageenan/TiO₂ beads crosslinked by mixed salt of KCl and CaCl₂ have higher adsorption capacity, as compared to beads crosslinked with single salt of KCl. The kinetic study indicated the chemisorption of Methylene Blue on the bead surface. κ-carrageenan/TiO₂ beads showed higher degradation for Methylene Blue and Nile Blue A than Rhodamine B in 5 and 50 ppm each multicomponent dye systems under UV irradiation. The κ-carrageenan/TiO₂ beads could form a porous column for the selective adsorption of positively charged dyes from a multicomponent dye system.     

Controlled release of aspirin from pH‐sensitive chitosan/poly(vinyl alcohol) hydrogel

The aim of this study was to develop a cheap, pH‐sensitive enteric coating of aspirin with biocompatible polymers. A novel approach was used to develop enteric coating from chitosan (CS) and poly(vinyl alcohol) (PVA). Solutions of CS and PVA (5 : 1 mol ratio) were mixed and selectively crosslinked with tetraethoxysilane. IR analysis confirmed the presence of the incorporated components and the existence of siloxane linkages between CS and PVA. The crosslinking percentage and thermal stability increased with increasing amount of crosslinker. The response of the developed coating in different media, such as water, pH (nonbuffer and buffer), and ionic media showed hydrogel properties. All hydrogels showed low swelling in acidic and basic pH media, whereas maximum swelling was exhibited at neutral pH. This pH sensitivity of the hydrogel has been exploited as enteric coating for commercial aspirin tablets. The dissolution test of enteric‐coated aspirin tablet in simulated gastric fluid (pH 1.2) showed 7.11% aspirin release over a period of 2 h, whereas a sustained release of remaining aspirin (83.25%) was observed in simulated intestinal fluid (pH 6.8). © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Low molecular weight water‐soluble chitosans: Preparation with the aid of cellulase,characterization, and solubility

Preparation of water‐soluble chitosan (WSC) was made by treating partially N‐deacetylated chitosan with acetic anhydride in aqueous acetic acid. The optimal conditions of preparing WSC were determined on the basis of orthogonal tests. Low molecular weight WSC with broad molecular weight (600–1.5 kDa) were obtained by the depolymerization of WSC using cellulase at optimum condition of pH 4.5 and 60°C. The solubility of WSC in water and aqueous organic solvents was investigated in detail. Weight–average molecular weight (M_w) and molecular weight distribution (M_w/M_n) of samples were measured by gel permeation chromatography. The structure of WSC and its degraded products were characterized by XRD, FTIR, and MALDI‐TOF MS. The decrease of molecular weight led to transformation of crystal structure and the increase of solubility, but the chemical structures of residues were not modified compared to WSC, which was not hydrolyzed. The solubility of the WSC in water and aqueous organic solvents increased with the decrease of molecular weight. The solubility of the WSC with low molecular weight was rather high even in aqueous dimethylacetamide and dimethylsulfoxide. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 1098–1105, 2006     

Mn(Ⅱ)对壳聚糖降解制备低聚壳聚糖的影响

将壳聚糖进行液态均相配合反应制得壳聚糖锰配合物，IR、元素分析及热分析等检测证实了壳聚糖锰配合物中配位键的存在，且显示壳聚糖锰配合物存在有利于壳聚糖高分子链断裂的弱势结构。以H2O2对壳聚糖．Mn(Ⅱ)配合物及壳聚糖进行氧化降解，考察降解过程中粘度的变化及降解产物分子量分布，在相同的降解条件下，壳聚糖锰配合物的降解速度明显高于壳聚糖，且降解产物分子量分布较壳聚糖直接降解窄。对壳聚糖锰配合物降解反应动力学研究表明壳聚糖锰配合物对H2O2分解不存在催化作用，其降解反应与壳聚糖的差异只与其结构有关。对降解产物进行脱金属处理，所得低聚壳聚糖含锰量为0。     

Polyelectrolyte complex hydrogel composed of chitosan and poly(γ‐glutamic acid) for biological application: Preparation,physical properties,and cytocompatibility

Polyelectrolyte complex (PEC) hydrogels composed of chitosan as a cationic polyelectrolyte and poly (γ‐glutamic acid) (γ‐PGA) as an anionic polyelectrolyte were prepared from PEC dispersions based on a chitosan solution to which different amounts of γ‐PGA solutions were added to charge equivalency. The chemical structures of the PEC hydrogels were investigated by Fourier transform infrared spectroscopy. The physical properties, fixed charge concentration, crystallinity, mechanical properties, micromorphology, and swelling properties of the PEC hydrogels were also investigated. The total fixed charge concentration of the PEC hydrogels varied as a function of pH on the pK intervals between chitosan (pK = 6.5) and γ‐PGA (pK = 2.27). The isoelectric points (IEP) were shifted to a lower pH with a higher weight ratio of γ‐PGA to chitosan. The elastic modulus was decreased with the weight ratio increasing from 0 : 1 to 1 : 1 (γ‐PGA/chitosan) by ionic crosslinking between the amino groups of chitosan and the carboxyl groups of γ‐PGA. The results of the swelling study showed that the swelling properties of PEC hydrogels were more affected by the change in the elastic restoring force than by the change in the fixed charge concentration depending on the pH. Also, the cytotoxicity of the PEC hydrogels was investigated using normal human dermal fibroblast (NHDF) cell lines, and the results showed the PEC hydrogels were not toxic. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103:386–394, 2007     

Effect of preparation method on the capture and release of biologically active molecules in chitosan gel beads

Model bioactive compounds with different molecular and functional characteristics were entrapped in gel beads prepared by polyelectrolyte complexation of chitosan and pentasodium tripolyphosphate (TPP). Three compounds of interest to the food and biomedical industries were tested: (1) lysine, (2) bovine serum albumin (BSA), and (3) β‐galactosidase. Effects of the compound concentration in the initial chitosan solution, pH of the curing solution, and length of the curing phase on the capture efficiency were evaluated. Release rates for lysine and BSA into a phosphate buffer, distilled water, and a synthetic ocean solution were observed, and the activity of the entrapped β‐galactosidase was determined. The capture efficiencies for lysine and BSA decreased as the concentration increased. The capture efficiency for lysine ranged from 90% at pH 5 to 20% at pH 8.6. There was no significant effect of the release media on the rate of release. BSA and lysine release reached 90% of the maximum after 100 and 50 min, respectively. The capture efficiency of β‐galactosidase was not affected by the pH of TPP; however, enzyme activity in the beads decreased as the pH increased. Beads prepared in the pH 8.6 TPP solution had significantly higher rates of enzyme release over time. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 405–413, 2002; DOI 10.1002/app.10393     

Degradation behavior of hydrogels from poly(vinyl alcohol)‐graft‐[poly(rac‐lactide)/poly(rac‐lactide‐co‐glycolide)]: Influence of the structure and composition on the material's stability

The swellability and mass loss of poly(vinyl alcohol)‐graft‐[poly(rac‐lactide)/poly(rac‐lactide‐co‐glycolide)] hydrogels upon hydrolysis are strongly affected by the composition of the hydrogels. Hydrophobic hydrogels remain at a relatively constant mass for a couple of weeks, and the mass decreases dramatically thereafter, whereas more hydrophilic hydrogels lose mass right from the beginning. All hydrogels display water uptake values between 90 and 280% within 8 weeks. For longer periods of degradation, the water uptake increases up to a maximum of about 900%. Studies of the thermal properties of samples upon degradation and their IR measurements have shown that the degradation rate is related to the physical and chemical structure of the hydrogels and hence to the hydrophobic/hydrophilic balance; that is, the degradation increases with the increasing hydrophilicity of the material. As a result, degradation can be engineered through the variation of the composition and structure of the material. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Encapsulation of salicylic acid in acylated low molecular weight chitosan for sustained release topical application

Acylated low molecular weight chitosan was used to encapsulate salicylic acid (SA) for sustained release in topical delivery. Chitosan nanoparticles were prepared from the depolymerization of commercial chitosan and further acylated with short alkyl chains. The successful acylation of butyryl chitosan [low molecular weight chitosan (LMWC)‐B] were proved by Fourier transform infrared (FTIR) and ¹H‐NMR. Successful encapsulation of SA was observed by the shift of amide I band from 1648 cm^?1 in LMWC‐B to 1641–1633 cm^?1 in SA‐loaded LMWC‐B in FTIR analysis, which further confirmed with the size increment from dynamic light scattering and transmission electron microscopy analyses by comparing its unencapsulated LMWC‐B. SA release from LMWC‐B studied by Franz diffusion experiment followed Korsmeyer–Peppas model where the release component n value (0.502) indicated diffusion and polymer swelling were involved in release mechanism. The slow release study of SA showed the acylated chitosan exhibited sustained release property toward SA. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 45273.     

壳聚糖黏均分子量对其作表面施胶剂应用效果的影响

利用过氧化氢对壳聚糖进行氧化降解,获得了一系列不同黏均分子量的壳聚糖。用不同黏均分子量的壳聚糖对纸张表面进行施胶,研究施胶对纸张性能的影响。结果表明:壳聚糖黏均分子量的降低不利于其表面施胶效果,并通过扫描电镜图进行了证实;在相同条件下,相比于单独使用氧化淀粉,少量壳聚糖与氧化淀粉共混作用于纸张表面,对纸张表面强度增强效果更佳。     

Properties and application of poly(methacrylic acid‐co‐dodecyl methacrylate‐cl‐N,N‐methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetate

A range of fatty acid esters is now being produced commercially with immobilized microbial lipases (glycerol ester hydrolases; EC) in nonaqueous solvents. In this study, a synthetic hydrogel was prepared by the copolymerization of methacrylic acid and dodecyl methacrylate in the presence of a crosslinker, N,N‐methylene bisacrylamide. A purified alkaline thermotolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a poly(methacrylic acid‐co‐dodecyl methacrylate‐cl‐N,N‐methylene bisacrylamide) hydrogel by an adsorption method. The hydrogel showed a 95% binding efficiency for the lipase. The bound lipase was evaluated for its hydrolytic potential toward various p‐nitrophenyl acyl esters with various C chain lengths. The bound lipase showed optimal hydrolytic activity toward p‐nitrophenyl palmitate at a pH of 8.5 and a temperature of 55°C. The hydrolytic activity of the hydrogel‐bound lipase was enhanced by Hg²⁺, Fe³⁺, and NH ions at a concentration of 1 mM. The hydrogel‐bound lipase was used to synthesize geranyl acetate from geraniol and acetic acid in n‐heptane. The optimization of the reaction conditions, such as catalyst loading, effect of substrate concentration, solvent (n‐pentane, n‐hexane, n‐heptane, n‐octane, and n‐nonane), reaction time, temperature, molecular sieve (3 Å × 1.5 mm) and scale up (at 50‐mL level), was studied. The immobilized lipase (25 mg/mL) was used to perform an esterification in n‐alkane(s) that resulted in the synthesis of approximately 82.8 mM geranyl acetate at 55°C in n‐heptane under continuous shaking (160 rpm) after 15 h when geraniol and acetic acid were used in a ratio of 100 : 100 mM. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 40 mg/mL in reaction volume (2 mL) resulted in an increase in the conversion of reactants into geranyl acetate (90.0 mM). During the repetitive esterification under optimum conditions, the hydrogel‐bound lipase produced ester (37.0 mM) after the eighth cycle of reuse. When the reaction volume was scaled up to 50 mL, the ester synthesized was 58.7 mM under optimized conditions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Removal of alkylphenols by the combined use of tyrosinase immobilized on ion‐exchange resins and chitosan beads

Mushroom tyrosinase was covalently immobilized on a poly(acrylic acid)‐type, weakly acidic cation‐exchange resin (Daiaion WK10, Mitsubishi Chemical Corp., Tokyo, Japan) with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride salt as a water‐soluble carbodiimide. Ion‐exchange resins immobilized with tyrosinase were packed in one column, and crosslinked chitosan beads prepared with epichlorohydrin were packed in another column. The enzymatic activity was modified by covalent immobilization, and the immobilized tyrosinase had a high activity in the temperature range of 30–45°C and in the pH range of 7–10. When solutions of various alkylphenols were circulated through the two columns packed with tyrosinase‐immobilized ion‐exchange resins and crosslinked chitosan beads at 45°C and pH 7 (the optimum conditions determined for p‐cresol), alkylphenols were effectively removed through quinone oxidation with immobilized tyrosinase and subsequent quinone adsorption on chitosan beads. The use of chemically crosslinked chitosan beads in place of commercially available chitosan beads was effective in removing alkylphenols from aqueous solutions in shorter treatment times. The removal efficiency increased with an increase in the amount of crosslinked chitosan beads packed in the column because the rate of quinone adsorption became higher than the rate of enzymatic quinone generation. The activity of tyrosinase was iteratively used by covalent immobilization on ion‐exchange resins. One of the most important findings obtained in this study is the fact that linear and branched alkylphenols suspected of weak endocrine‐disrupting effects were effectively removed from aqueous solutions. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010