Covalent Immobilization of Lipase on Poly（ acrylonitrile-co-maleic acid） Ultrafiltration Hollow Fiber Membrane

IntroductionLipases are biotechnologically important enzymes,which are able to catalyze the hydrolysis/synthesis of awide range of soluble or insoluble carboxylic acid estersand amides.In this way,the enzymes have been wide-ly used biotechnologically in dairy industry,oil pro-cessing,the production of surfactants,and the prepara-tion of enantiomerically pure pharmaceuticals[1,2].However,like mostenzymes for industrial applica-tions,lipases are unstable and easy to lose their cata-lytic activit…     

Catalytic activity of lipase immobilized onto ultrathin films of cellulose esters

Ultrathin (approximately 2.0 nm) films of cellulose acetate (CA), cellulose acetate propionate (CAP), and cellulose acetate butyrate (CAB) supported on Si wafers have been prepared by adsorption and characterized by means of ellipsometry, atomic force microscopy (AFM), and contact angle measurements. CA, CAP, and CAB ultrathin films were characterized in air just after their formation and after annealing under reduced pressure at temperature higher than the corresponding melt temperature. Upon annealing, CA, CAP, and CAB ultrathin films became smoother and more hydrophobic, evidencing molecular reorientation at the solid-air interface. CA, CAP, and CAB films were used as supports for the immobilization of lipase. The adsorption of lipase onto annealed films was more pronounced than that onto untreated films, showing the strong affinity of lipase for the more hydrophobic substrates. Enzymatic activity was evaluated by a standard procedure, namely, (spectrophotometric) measurement of p-nitrophenol, the product formed from the hydrolysis of p-nitrophenyl dodecanoate (p-NPD). Lipase immobilized onto hydrophobic films exhibited higher activity than that of free lipase and could be recycled three times while retaining relatively high activity (loss of ca. 30% of original enzymatic activity). The effect of storing time on the activity of immobilized lipase was studied. Compared with free lipase, that immobilized onto more hydrophobic films retained 70% activity after 1 month. More importantly, the latter level of activity is similar to that of free lipase. However, lipase immobilized onto more hydrophilic films retained 50% and 30% activity after 20 and 30 days, respectively. These results are explained in terms of surface wettability and the contribution of the interactions between the polar residues of lipase and the glucopyranosyl moieties of cellulose ester to maintain the natural conformation of immobilized enzyme.     

Method for grafting poly(acrylic acid) onto nylon 6,6 using amine end groups on nylon surface

Several methods have been developed for grafting materials to the surface of polymers to alter their surface characteristics. This article reports a procedure for grafting poly(acrylic acid) (PAA) onto nylon 6,6 films via the naturally occurring amine end groups of nylon 6,6 using N‐hydroxy‐succinimide in conjunction with 1‐ethyl‐3‐ (3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) facilitated amidazation. Reaction conditions were investigated with respect to PAA molecular weight, activator concentrations, reaction temperature, and time. X‐ray photoelectron spectroscopy showed that surface coverage of more than 50% was consistently achieved for 250 kD PAA. The maximum grafting occurred at room temperature with a large excess of EDC with a reaction time of 30 min. The same level of grafting can be achieved using smaller amounts of EDC at 60 °C. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 719–728, 2002; DOI 10.1002/pola.10149     

Surface methacrylation and graft copolymerization of ultrafine cellulose fibers

Ultrafine fibrous (? from 100 to 450 nm) cellulose membranes were generated by electrospinning of cellulose acetate [degree of substitution (DS): 2.45, weight‐average molecular weight: 30,000 Da], followed by alkaline deacetylation. Reaction of these ultrahigh surface‐area cellulose fibers with methacrylate chloride (MACl) produced activated surfaces without altering the fiber morphology. Surface methacrylation of these fibers was confirmed by the acquired hydrophobicity (θ_water = 84°) as compared to the originally hydrophilic (θ_water = 56°) cellulose. Changing the MACl:OH molar ratios could vary the overall DS of methacrylation. The very low overall DS values indicate the surface nature of the methacrylation reaction. At a DS of 0.17, the thermal properties of the surface methacrylated cellulose resemble those of cellulose derivatives at much higher DS values, an unusual behavior of the ultrafine fibers. The methacrylated cellulose could be further copolymerized with vinyl monomers (methyl methacrylate, acrylamide, and N‐isopropylacrylamide) as linear grafts or three‐dimensional (3D) networks. The morphology of cellulose fibers and the interfiber pore structure were not altered at 15–33% graft levels. This study demonstrates that either linear or 3D networks of vinyl polymers could be efficiently supported on ultrafine cellulose fibrous membranes via surface methacrylation. Through these surface reactions the chemical, thermal, and liquid wetting and absorbent properties of these ultrafine fibrous membranes were significantly altered with no change to the fiber dimensions or interfiber pore morphology. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 953–964, 2003     

Characterization of carbonyl chloride-terminated aromatic polyamide nanoparticles with carboxyl groups and their reaction

Aromatic polyamide nanoparticles with carbonyl chloride (COCl) and carboxyl (COOH) groups were obtained using a precipitation polymerization method. The morphology, number of COCl groups incorporated, and degree of polymerization of the resulting particles depended on the reaction system. The COOH group of diamine used decreased the reactivity of NH₂ groups and chemically stabilized the COCl groups existing at the ends of the molecular chains. Also, the COCl groups were retained in particles by the rapid formation of particles. Thus, the chemical structures and formation mechanism were found to play an important role in the formation of particles with COCl groups.     

Effect of surface‐grafted ionic groups on the performance of cellulose‐fiber‐reinforced thermoplastic composites

The influence of the surface chemistry of the cellulose fiber and polymer matrix on the mechanical and thermal dynamic mechanical properties of cellulose‐fiber‐reinforced polymer composites was investigated. The cellulose fiber was treated either with a coupling agent or with a coupling‐agent treatment followed by the introduction of quaternary ammonium groups onto the fiber surface, whereas the polymer matrix, with opposite polar groups such as polystyrene incorporated with sulfonated polystyrene and poly(ethylene‐co‐methacrylic acid), was compounded with the fiber. The grafting of the fiber surface was investigated with Fourier transform infrared spectroscopy and X‐ray photoelectron spectroscopy. Experimental results showed that an obvious improvement in the mechanical strength could be achieved for composites with an ionic interface between the fiber and the polymer matrix because of the adhesion enhancement of the fiber and the matrix. The improved adhesion could be ascribed to the grafted ionic groups at the cellulose‐fiber surface. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2022–2032, 2003     

Hydrolysis of 1,2-diacetoxypropane by Immobilized Lipase on Cellulose Acetate-TiO2 Gel Fiber Derived from the Sol-Gel Method

Lipase from Rhizomucor miehei was entrap-immobilized on cellulose acetate-TiO₂ gel fiber by the sol-gel method. This fiber-immobilized lipase was stable in a phosphate buffer solution and easy to handle. The enantioselective hydrolysis of 1,2-diacetoxypropane catalyzed by this immobilized lipase could be performed in buffer solution unlike the lipase immobilized on an alginate matrices. The enantioselectivity was improved in presence of this fiber-immobilized lipase compared with the hydrolysis catalyzed by the native lipase. This finding indicates that the active site structure of lipase immobilized on fiber was retained to some extent, though the enzyme conformation may become flexible in presence of water. We also compared the properties of this fiber-immobilized lipase with native lipase and commercially available immobilized lipase from Rhizomucor miehei, viz., Lipozyme.     

PEG–PLGA copolymers bearing carboxylated side chains: Novel hydrogels with enhanced crosslinking via ionic interactions

Novel water‐soluble amphiphilic block copolymers with pendant carboxylic acid groups are synthesized and used for the preparation of ionically crosslinked hydrogels. d ,l ‐Lactide (DLLA) and l ?3‐(2‐benzyloxycarbonyl)ethyl‐1,4‐dioxane‐2,5‐dione (BED) are copolymerized at different ratios via organo‐catalyzed ring‐opening polymerization using a hydroxyl‐terminated poly(ethylene glycol) (PEG–OH) macroinitiator. Dynamic light‐scattering experiments show that, at low concentrations, aqueous solutions of these PEG‐P(BED‐DLLA) copolymers form micelles and aggregates. At higher concentrations, thermo‐sensitive gels are obtained, exhibiting a reversible gel‐to‐sol transition upon a temperature increase. Ionic interactions between the COOH groups and metal ions (Cu²⁺ or Ca²⁺) are shown to significantly shift the gel–sol transition to higher temperatures. Thus, the introduction of COOH groups significantly enhances the water solubility of the amphiphilic PEG–polyester copolymer and allows additional crosslinking interactions to form functionalized hydrogels with improved physical properties, making this new class of hydrogels interesting for various applications. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1222–1227     

Synthesis,characterization, and stability of carbodiimide groups in carbodiimide‐functionalized latex dispersions and films

Cyclohexylcarbodiimidoethyl methacrylate (CCEMA) and t‐butylcarbodiimidoethyl methacrylate (t‐BCEMA) were prepared in a two‐step synthesis. These monomers were then used to prepare carbodiimide‐functionalized PBMA and PEHMA latex particles, employing two‐stage emulsion polymerization, with the carbodiimide–methacrylate monomers being introduced only in the second stage under monomer‐starved conditions. During emulsion polymerization, the carbodiimide moiety ( NCN ) was found to be unstable at pH 4, but stable when the pH of the dispersion was increased to 8, using NaHCO₃ as the buffer. Survival of  NCN group against hydrolysis during the polymerization, and during storage in the dispersion, was enhanced by using EHMA as the comonomer (more hydrophobic) and the t‐butyl carbodiimide derivative. The t‐butyl group provides more steric hindrance to the hydrolysis reaction. A decrease in the reaction temperature from 80°C to 60°C was also found to increase the extent of  NCN group incorporation during emulsion polymerization. Under ideal conditions, more than 98% of the  NCN groups in the monomer feed are successfully incorporated into the latex. When these latex particles are mixed with a  COOH containing latex and allowed to dry, polymer diffusion leading to crosslinking occurs. Films annealed at 60°C reach a gel content of 60% in 10 h. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 855–869, 2000     

Synthesis and degradation of end‐group‐functionalized polylactide

Three kinds of OH‐terminated polylactides were synthesized by the ring‐opening polymerization of lactide, with an alcohol such as dodecanol, glycerol, or pentaerythritol, in the presence of stannous octoate. Moreover, Cl‐, NH₂‐, and COOH‐terminated polylactides were synthesized from OH‐terminated polylactides. The end groups of the polylactides were identified by ¹H NMR and ¹³C NMR. According to thermal analysis, the cold crystallization temperatures of Cl‐, NH₂‐, and COOH‐terminated polylactides were higher than those of OH‐terminated polylactides. The thermal stability of OH‐terminated polylactides was poor, whereas NH₂‐ and Cl‐terminated polylactides were more resistant to thermal degradation. In a hydrolysis degradation test, the mass and molecular weight loss of COOH‐terminated polylactides were high, whereas those of Cl‐ and NH₂‐terminated polylactides were much lower. These end‐group effects were increased with an increasing number of chain arms. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 973–985, 2001     

Lipase bound cellulose nanofibrous membrane via Cibacron Blue F3GA affinity ligand

Cellulose (Cell) nanofibrous membranes were prepared by nucleophilic reaction of the cellulose hydroxyl with the triazinyl chloride of Cibacron Blue F3GA (CB) ligand and studied as affinity membranes for lipase enzyme. Cell nanofibrous membranes containing fibers with 200 nm average diameters were prepared by electrospinning of cellulose acetate (CA), followed by alkaline hydrolysis. The CB capacity of the Cell nanofibrous membranes was optimized by lengthening the nucleophilic reaction time and increasing CB concentration and ionic strength. The equilibrium adsorption isotherms of CB on the Cell nanofibrous membranes followed a typical Langmuir monolayer adsorption behavior. At 242 mg CB/g of Cell, the maximum lipase adsorption capacity (q_m) and the dissociation constant (K_d) values were 41.02 mg/g and 0.25 mg/mL, respectively. Optimal lipase adsorption capacity was obtained at pH 4.0, its isoelectric point, with added NaCl on Cell membranes 86 mg CB capacity per g of Cell. A facile lipase loading capacity of 16.21 mg/g of CB–Cell was achieved under moderated conditions and could be optimized to reach at least 150 mg/g. The CB–Cell bound lipase had similar catalytic rate and retained 86.2% activity as in its free form. These findings clearly show that the CB bound Cell nanofibrous membrane is a highly efficient ultra-high specific porous support for lipase enzyme and is potentially versatile for immobilizing other enzymes and as affinity membrane for proteins.     

Physical properties of glycosaminoglycan hydrogels

The chemical composition of glycosaminoglycan (GAG) hydrogels was found to have a profound effect on the physical properties of gels. Hyaluronan (HA) and chondroitin sulfate (CS) were each modified with adipic dihydrazide (ADH) with carbodiimide chemistry. The resulting polymer was crosslinked with various concentrations of poly(ethylene glycol) dialdehyde (PEG‐diald) to produce a series of hydrogels. The physical properties of these GAG hydrogels varied in a concentration‐dependent fashion. Maximal crosslinking was observed at a theoretical crosslinking of 50% for the HA‐ADH‐PEG‐diald hydrogels and 75% for the CS‐ADH‐PEG‐diald hydrogels. Adding PEG‐diald beyond the optimum for crosslinking prolonged the in vitro enzymatic degradation time of the hydrogels. The swelling of the crosslinked GAG hydrogels was correlated with the amount of PEG‐diald used rather than with the crosslinking density. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 4344–4356, 2004     

Synthesis and properties of ultralow dielectric porous polyimide films containing adamantane

A series of novel ultralow dielectric porous polyimide (PI) films containing adamantane groups was prepared via the thermolysis of polyethylene glycol (PEG) oligomers mixed into PI matrix. Scanning electron microscopy results indicated that the porous PI films showed closed pores with an average diameter of 120 ± 10 nm. Good thermal properties with 5% weight loss temperature of 499 °C in air atmosphere and glass transition temperature in excess of 310 °C were shown for porous PI films. Notably, the ultralow dielectric constant of porous PI films with 1.85 at 1 MHz was obtained and revealed via broadband dielectric spectroscopy. The effects of the chemical structure of the PI matrix and PEG content on the decomposition behavior of PEG and the performance of porous films were investigated. Wide‐angle X‐ray diffraction results indicated that the PI matrix with large d‐spacing generated weaker interactions between the PEG and PI backbone than those of PI matrix with small d‐spacing. As a result, the PEG for the PI matrix with large d‐spacing was completely decomposed. As indicated by the broadband dielectric spectroscopy results, lower dielectric porous PI films were prepared when the PEG contents in the PI matrix increased from 0 to 20 wt %. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 549–559     

Synthesis and characterization of polyphenylenes with polypeptide and poly(ethylene glycol) side chains

We report a novel approach for fabrication of multifunctional conjugated polymers, namely poly(p‐phenylene)s (PPPs) possessing polypeptide (poly‐l ‐lysine, PLL) and hydrophilic poly(ethylene glycol) (PEG) side chains. The approach is comprised of the combination of Suzuki coupling and in situ N‐carboxyanhydride (NCA) ring‐opening polymerization (ROP) processes. First, polypeptide macromonomer was prepared by ROP of the corresponding NCA precursor using (2,5‐dibromophenyl)methanamine as an initiator. Suzuki coupling reaction of the obtained polypeptide and PEG macromonomers both having dibromobenzene end functionality using 1,4‐benzenediboronic acid as the coupling partner in the presence of palladium catalyst gave the desired polymer. A different sequence of the same procedure was also employed to yield polymer with essentially identical structure. In the reverse sequence mode, low molar mass monomer (2,5‐dibromophenyl)methanamine, and PEG macromonomer were coupled with 1,4‐benzenediboronic acid in a similar way followed by ROP of the L‐Lysine NCA precursor through the primary amino groups of the resulting polyphenylene. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1785–1793     

Beyond PEG2000: Synthesis and Functionalisation of Monodisperse PEGylated Homostars and Clickable Bivalent Polyethyleneglycols

A new strategy to access highly monodisperse, heterobifunctional linear polyethylenglycols (PEGs) has been designed. This was built around unidirectional, iterative chain extension of a 3‐arm PEG homostar. A mono‐(4,4′‐dimethoxytriphenylmethyl) octagol building block, DmtrO‐EG₈‐OH, was constructed from tetragol. After six rounds of chain extension, the monodisperse homostar reached the unprecedented length of 56 monomers per arm (PEG2500). The unique architecture of the synthetic platform greatly assisted in facilitating and monitoring reaction completion, overcoming kinetic limitations, chromatographic purification of intermediates, and analytical assays. After chain terminal derivatisation, mild hydrogenolytic cleavage of the homostar hub provided heterobifunctional linear EG₅₆ chains with a hydroxyl at one end, and either a toluene sulfonate, or a tert‐butyl carboxylate ester at the other. A range of heterobifunctional, monodisperse PEGs was then prepared having useful cross‐linking functionalities (‐OH, ‐COOH, ‐NH₂, ‐N₃) at both ends. A rapid preparation of polydisperse PEG homostars, free of multiply cross‐linked chains, is also described. The above approach should be extendable to other high value oligomers and polymers.     

One‐step synthesis of polycarbonates bearing pendant carboxyl groups by lipase‐catalyzed ring‐opening polymerization

The ring‐opening polymerization of a monomer containing a free carboxylic acid group is reported for the first time. The monomer, 5‐methyl‐5‐carboxyl‐1,3‐dioxan‐2‐one (MCC), was copolymerized with trimethylene carbonate (TMC) in an enzymatic ring‐opening polymerization conducted in bulk at 80 °C. The low‐melting TMC comonomer also solubilized the high‐melting MCC monomer, allowing for solvent‐free polymerizations. Six commercially available lipases were screened, and Candida antarctica lipase‐B (Novozym‐435) and Pseudomonas cepacia lipase were selected to catalyze the copolymerization because of their higher monomer conversions. Higher molecular weight polymers (weight‐average molecular weight = 7800–9200) were prepared when Novozym‐435 was used, with less MCC incorporated into the copolymer than used in the monomer feed. However, Pseudomonas cepacia lipase showed good agreement between the molar feed ratios and the molar composition, but the molecular weights (weight‐average molecular weight = 3600–4800) were lower than those obtained when Novozym‐435 was used. ¹³C NMR spectral data were used for microstructural analysis, which suggested the formation of random, linear, and pendant carboxylic acid groups containing polycarbonates with hydroxyl groups at both chain ends. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1267–1274, 2002     

界面活化的溶胶凝胶包埋Candida rugosa脂肪酶催化合成维生素E琥珀酸酯

采用界面活化的溶胶凝胶包埋Candida rugosa脂肪酶(CRL)催化合成了维生素E琥珀酸酯.考察了影响溶胶凝胶包埋固定化CRL的因素,获得的最佳固定化条件为:丙基三甲氧基硅烷/正硅酸四乙酯摩尔比为1/1,水与硅烷前体摩尔比为15,酶的添加量为0.5mg/ml,PEG400的添加量为12μl/ml溶胶. 溶胶凝胶包埋的CRL在50℃,18h后其活性仍然保持了70.58%,是游离酶的2.6倍,且稳定性得到了明显的改善.基于CRL的界面特性,采用五种表面活性剂对其进行界面活化.结果表明,采用橄榄油活化的溶胶凝胶包埋的CRL合成维生素E琥珀酸酯的酯化活力最高,相比原酶和未界面活化的溶胶凝胶包埋酶分别提高了6.7和1.43倍.     

Synthesis of a “molecular rope curtain”: Preparation and characterization of a sliding graft copolymer with grafted poly(ethylene glycol) side chains by the “grafting onto” strategy

A sliding graft copolymer (SGC) with poly(ethylene glycol) (PEG) side chains was prepared by ester formation between terminal carboxyl groups of oxidized PEG methyl ether with molecular weight of 2000 (mPEG2000‐COOH) and hydroxyl groups of a polyrotaxane consisting of PEG and cyclodextrins (CDs). Formation of the SGC structure was confirmed by ¹H NMR, attenuated total reflectance Fourier‐transformed infrared, and gel permeation chromatography. The SGC was soluble in good solvents of PEG and insoluble in poor solvents of PEG. Estimation of the number of grafted mPEG chains suggested a “rope‐curtain” like structure, in which an mPEG chain is connected to each CD ring. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011