Some new reactive polymers for the immobilization of enzymes.
From poly(vinyl alcohol) precursors, various reactive carriers for the immobilization of enzymes were synthesized. As insoluble starting polymers, the following products were used: poly(vinyl alcohol), gels crosslinked with terephthalaldehyde, hydrolyzed beads of crosslinked poly(vinyl acetate), poly(vinyl acetate-co- ethylene) tubes coated with poly(vinyl alcohol), and poly(vinyl alcohol)-containing synthetic pulp. Reactive groups introduced into these carriers or methods for their activation included the diazonium- and isothiocyanato group, and the glutardialdehyde-, BrCN, 2, 4, 6-trichloro-s-triazien, and p-benzoquinone methods. Furthermore, SH-specific reactive groups such as N-substituted maleimide groups or activated mixed disulfides with 2-thiopyridyl groups could be introduced into PVA-polymers. Enzymes like hydrolases (e.g. papain, trypsin, chymotrypsin, urease), oxidoreductases (e.g. glucose oxydase, catalase, glucose-6-phosphate dehydrogenase) as well as the example of transferase hexokinase coimmobilized with glucose-6-phosphate dehydrogenase, were immobilized by reactive poly(vinyl alcohol) carriers. The properties of the immobilized enzymes were investigated.
Properties and enzymatic activities of papain insolubilized with glutaraldehyde.
Abstract Glutaraldehyde at a concentration of 2.3% and at 0 ° reacted in aqueous solutions with papain to form a water-insoluble product with enzymatic activity after activation by reducing agents. A rapid reaction of glutaraldehyde with the essential sulfhydryl of papain was not involved in the reaction since after activation the insoluble enzyme retained esterolytic and proteolytic activity. The amount of enzymatic activity retained by the insoluble material depended on the conditions of the reaction. The insolubilizatiou of papain with glutaraldehyde was pH dependent; the higher the pH the more rapid the reaction. Complete precipitation occurred in 24 hr over the range pH 5.2–7.2. The lower the pH of the reaction, the greater was the retention of esterase activities. Similar results were obtained when the reaction was carried out in the presence of added cysteine. However, the insoluble enzyme prepared in the absence of cysteine was fibrous while that prepared in its presence was crystalline. The reaction of mercuripapain with glutaraldehyde resulted in an insoluble enzyme that possessed, after treatment with cysteine, approximately twice as much esterase and proteinase activities as did the insoluble enzyme prepared from papain. Compared with soluble papain, preparations from glutaraldehyde mercuri-papain retained 48% of the ester, 12% of the casein, and 16% of the hemoglobin hydrolyzing ability. The reaction of the reduced soluble and insoluble enzymes with iodoacetamide-1- 14 C demonstrated that insoluble mercuripapain retained all of its essential sulfhydryl groups whereas those in insoluble papain had been reduced to two thirds. Amino acid analysis on hydrolyzed insoluble enzyme showed that only the lysine had been significantly altered by the glutaraldehyde reaction, having been reduced to approximately one-half that of soluble papain under all the conditions studied.
Engineering bio-inks for 3D bioprinting cell mechanical microenvironment
Three-dimensional (3D) bioprinting has become a promising approach to construct­ing functional biomimetic tissues for tissue engineering and regenerative medicine. In 3D bioprinting, bio-inks are essential for the construction of cell microenviron­ment, thus affecting the biomimetic design and regenerative efficiency. Mechanical properties are one of the essential aspects of microenvironment, which can be char­acterized by matrix stiffness, viscoelasticity, topography, and dynamic mechanical stimulation. With the recent advances in functional biomaterials, various engineered bio-inks have realized the possibility of engineering cell mechanical microenviron­ment in vivo. In this review, we summarize the critical mechanical cues of cell micro­environments, review the engineered bio-inks while focusing on the selection princi­ples for constructing cell mechanical microenvironments, and discuss the challenges facing this field and the possible solutions for them.