Encapsulate trypsin

Trypsin-encapsulated sol-gel (alkoxysilane-based) was fabricated in situ onto the sample reservoir of a PMMA chip. This was employed for enzymatic conversion of NBD-labeled ArgOEt and bradykinin, followed by CE separation of the products. The enzymatic activity of the encapsulated trypsin as given by the Km value was found to be 19 times higher than that of the free trypsin. The stability of trypsin was 1 week at 4°C. This enhanced enzyme stability was possibly caused by the prevention of enzyme autolysis by the sol-gel matrix [1062],... 

Sakai-Kato, K., Kato, M., Toyo oka, T., Creation of an on-chip enzyme reactor by encapsulating trypsin in sol-gel on a plastic microchip. Anal. Chem. 2003, 75, 388-393. 

There are also examples of nanoreactors which have increased enzyme activity when encapsulated compared with bulk conditions. PICsomes that are stable to salt and elevated temperatures have been successfully developed using PEG-h-P( Asp) and are able to encapsulate trypsin without altering the original enzymatic activity [25]. Furthermore, these specifically designed PICsomes maintained their vesicular architecture at salt concentrations of 300 mM NaCl and temperatures up to 70°C, with only a subtle inaease in their size. For example, encapsulated nucleoside hydrolase has a substrate turnover number (kat) up to six times higher than the free enzyme [59]. Other enzymes, such as GOx and HRP, showed an 85% inaease in activity afta encapsulation in polyelectrolyte capsules [112], In addition, laccase entrapped in linear-dendritic based nanoreactors showed superior activity compared with its activity in bulk, and also inaeased temperature stability (up to 70 C) [19]. Howeva, the intrinsic branched architecture of den-drimers offers insufficient fluidity and space for entrapping larger biomolecules. 

Kato KS, Kato M, Toyo Oka T (2003) Creation of an On-Chip Enzyme Reacte by Encapsulating Trypsin in Sol-Gel on a Plastic Microchip. Anal Chem 75 388-293... 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

Clnkotai (33) in 1976 reported that byssinosis correlates with the activity of trypsin-like enzymes in cardroom dust and with the concentration of 1-2 ym, and 2-4 ym particle fractions. These fractions contained the viable microorganisms carrying pro-tealytic enzymes. The enzymes were also present in wool mills (no byssinosis) but were considered to be encapsulated in microorganisms. ... 

Sakai-Kato K, Kato M, Toyo oka T (2002) On-line trypsin-encapsulated enzyme reactor by the sol-gel method integrated into capillary electrophoresis. Anal Chem 74 2943-2949... 

Dendrimers have an architecture which is intrinsically accessible, because their branched structure allows the passage of substfates and products. In the case of PICsomes, the use of polyelectrolytes, which self-assemble into intrinsically porous systems, favors accessibility to the inner cavity where the active compounds are located [25]. Their membranes are permeable to small molecules, such as Tokyo-Green- 3-galactoside, while encapsulated active biomacromolecules, such as trypsin, cannot escape from the vesicular assemblies [25]. In the case of LbL capsules, permeabiUzation of the shell is tuned by its polymer composition, thickness, solvent, pH, or ionic strength [88,89]. 

Entrapment MicroChannel and stainless steel tubing fabricated using PDMS casting and 02-plasma treatment to generate a layer of silanol groups on the wall for covalent attachment via the hydroxyl groups of trypsin encapsulated in titania or alumina sol matrix digestion of BSA required 2 s reaction time... 

Entrapment Trypsin encapsulated sol-gel fabricated on sample reservoir of PMMA... 

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