• Journal of the Korean Society of Clothing and Textiles
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• v.37 no.7
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• pp.852-863
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• 2013

We investigate the immobilization of trypsin on chitosan nonwoven using glutaraldehyde (GA). The conditions for trypsin on chitosan nonwoven and GA cross-linking were optimized depending on different conditions. The order of GA cross-linking was determined by the activity of immobilized trypsin. The characteristics of chitosan nonwoven were examined by Fourier-transform infrared (FT-IR) and surface morphology analyses (SEM). Results showed that the optimal treatment conditions for trypsin on chitosan nonwoven were as follows: pH 8.5; temperature $37^{\circ}C$; trypsin concentration 15% (o.w.f); and treatment time 60 min. Those for GA cross-linking were: pH 10.0; GA concentration 3% (v/v); and treatment time 120 min. FT-IR analysis showed that GA was cross-linked on chitosan nonwoven. The SEM analysis also showed that trypsin was immobilized on chitosan nonwoven.

• Bulletin of the Korean Chemical Society
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• v.33 no.7
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• pp.2181-2186
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• 2012

Dendrimers are a novel class of nonlinear polymers and due to their extensive applications in different fields, called versatile polymers. Polyamidoamine (PAMAM) dendrimers are one of the most important dendrimers that have many applications in nanobiotechnology and industry. Generally aldehyde terminated dendrimers are prepared by activation of amine terminated dendrimers by glutaraldehyde which has two problems, toxicity and possibility of crosslink formation. In this study, novel aldehyde-terminated PAMAM dendrimer was prepared and used for covalent immobilization of trypsin by the aim of finding a special reagent which can prevent crosslinking and deactivation of the enzyme. For this purpose aminoacetaldehydedimethylacetal (AADA) was used as spacer group between aldehyde-terminated PAMAM and trypsin.The findings of this study showed that immobilization of trypsin not only resulted higher optimal temperature, but also increased the thermal stability of the immobilized enzyme in comparison to the free enzyme.

• KSBB Journal
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• v.21 no.4
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• pp.279-285
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• 2006

We investigated the effects of immobilization chemistry on the yield of immobilization and the bioactivity of the immobilized enzymes. Trypsin as a model protein and macroporous polymer beads(Toyopearl AF 650M, Tosho Co., Japan) was used as a model matrix. Four methods were used to immobilize trypsin; covalent conjugation by reductive amination(at pH 10.0 and pH 4.0) and affinity interaction via streptavidin-biotin, and double-affinity interaction via biotin-streptavidin-biotin system. The covalent conjugation immobilized $3{\sim}4$ mg/ml-gel, ca. 3-fold higher than the affinity method. However, the specific activity of the covalently(pH 10.0) and affinity-immobilized trypsin(via streptavidin-biotin) are ca. 37% and 50%, respectively, of that of the soluble enzyme(on the low-molecular-weight BAPNA substrate). When the molecular size of a substrate increased, the affinity-immobilized trypsin showed higher clavage activity on insulin and BSA. This result seemed to indicate the streptavidin-biotin system allowed more steric flexibility of the immobilized trypsin in its interaction with a substrate molecule. To confirm this, we studied the molecular flexibility of immobilized trypsin using quartz crystal microbalance-dissipation. Self-assembled monolayers were formed on the Q-sensor surface by aminoalkanethiols, and gultaraldehyde was attached to the SAMs. Trypsin was immobilized in two ways: reductive amination(at pH 10.0) and the streptavidin-biotin system. The dissipation shift of the affinity-immobilized trypsin was $0.8{\times}10^{-6}$, whereas that of the covalently attached enzyme was almost zero. This result confirmed that the streptavidin-biotin system allowed higher molecular flexibility. These results suggested that the bioactivity of the immobilized enzyme be strongly dependent on its molecular flexibility.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.2
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• pp.195-200
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• 2004

Trypsin can be immobilized on silk fibroin fiber (SFF) by introducing several spacer arms, such as ethylene diamine (ED), bovine serum albumin (BSA) and silk sericin (SS). Direct immobilization on silk fiber (SFFGA) has low activity because of the steric hindrance between the trypsin and substrate. The introduction of spacer arms onto SFF-GA can enhance the activity of trypsin by reducing the steric hindrance. When ED is used as a spacer arm, the activity of trypsin has increased but its stability decreased due to the increased hydrophobicity of SFF. BSA and SS, as a spacer arm, have better results in both activity and stability. SFF-BSA shows some decrease in the specific activity due to improper immobilizatin. SFF-SS maintained 90% of its initial activity even after 12 hrs incubation at $50^{\circ}C$. In the case of repeated hydrolysis of silk sericin with immobilized trypsin, SFF-GA and SFF-ED lost 50% of their initial activity right after first run, whereas SFF-BSA and SFF-SS maintained 80% of their initial activities even after 5 runs. Higher operational stability is due to increased hydrophilicity of SFF by introducing hydrophilic spacer arms such as BSA and SS. The high content of serine in SS increases the hydrophilicity of SFF resulting the best results among other spacer arms.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.35 no.2
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• pp.39-45
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• 2003

This study was performed to develop the new type enzyme immobilization sheet from carboxymethylated refiner mechanical pulp (CRMP) substrate. Enzyme immobilization was attempted to couple carboxyl groups of CRMP with amino groups of the enzyme, trypsin, through the reaction of carbodiimide reagent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodimide (EDC ). Immobilization carrier, water insoluble CRMP fraction (CRMP-IS), was successfully reacted with the enzyme, formed peptide linkage like -CONH- at 1680$cm^{-1}$ / and new ester linkage like -COO$CH_3$, methylester at 1735$cm^{-1}$ /, and produced enzyme immobilized substrate (CRMP-IST). The enzyme immobilized handsheet was prepared by mixing the above chelated enzyme immobilized substrate(CRMP-IST) with kraft pulp by paper sheet machine like papermaking process. The sheet weight and strength were increased with increasing dosage of CRMP-IST, and decreased at more than 10％ mixing of CRMP-IST, but higher than the controls. Concerning activities of immobilized trypsin(CRMP-IST) sheet by caseinolysis, the teared-off sheet with shaking was shown higher enzyme activities than sheet shape without shaking. In conclusion, this enzyme immobilized sheet would be expected easy handling for practical application and reutilization.

• Textile Coloration and Finishing
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• v.30 no.4
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• pp.264-274
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• 2018

Immobilization of lysozyme on chitosan non-woven using glutaraldehyde(GA) was investigated. For this, 100 % chitosan non-woven was prepared as novel support for the enzyme immobilization. In addition, free lysozyme activity was examined depending on various pH and temperature by measuring time. Moreover, the optimum immobilization conditions depending on various pH, temperature, immobilization time and lysozyme concentration was evaluated. In addition, thermal stability and storage stability of immobilized lysozyme were measured. The characteristics of immobilized lysozyme was examined by FT-IR, surface morphology, and MTT assay. The results are follows: the optimal immobilization of lysozyme were pH 7.0, $25^{\circ}C$, lysozyme concentration 1.5 mg/ml, immobilization time 240 min. The immobilized lysozyme showed higher thermal stability than the free trypsin. The immobilized lysozyme activity was retained 80 % of its initial activity at $4^{\circ}C$ over 30 days of storage. The lysozyme was immobilized effectively on chitosan non-woven by observation of surface morphology.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2004.04a
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• pp.23-24
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• 2004

• Bulletin of the Korean Chemical Society
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• v.33 no.10
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• pp.3233-3240
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• 2012

We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

• KSBB Journal
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• v.23 no.4
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• pp.350-354
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• 2008

Magnetically separable immobilized trypsin was developed and their biocatalytic activity was evaluated for the different immobilization media. The activity, recyclability, pH effect, and stability of immobilized enzymes were evaluated for the different supporting media. The biocatalytic activity of immobilized trypsin was highest with magnetically separable polyaniline (PAMP), and Vm and Km of PAMP were 0.169 mM/min and 0.263 mM respectively. With increasedpH, the biocatalytic activity increased for all supporting materials used. Immobilized enzymes were recycled and recycle activities were over 90% of their original activity after ten times reuse. The operational stabilities of enzymes were greatly improved with enzyme immobilization.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.4
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• pp.277-281
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• 2006

Spherical micro silica sol-gel immobilized enzyme beads were prepared in an emulsion system using cyclohexanone and Triton-X 114. The beads were used for the in situ immobilization of transaminase, trypsin, and lipase. Immobilization during the sol to gel phase transition was investigated to determine the effect of the emulsifying solvents, surfactants, and mixing process on the formation of spherical micro sol-gel enzyme beads and their catalytic activity. The different combinations of sol-gel precursors affected both activity and the stability of the enzymes, which suggests that each enzyme has a unique preference for the silica gel matrix dependent upon the characteristics of the precursors. The resulting enzyme-entrapped micronsized beads were characterized and utilized for several enzyme reaction cycles. These results indicated improved stability compared to the conventional crushed form silica sol-gel immobilized enzyme systems.