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Title: Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

Abstract

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.

Authors:
; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
898617
Report Number(s):
PNNL-SA-45554
24826; 24801; TRN: US200706%%231
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biotechnology and Bioenegineering, 96(2):210-218
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADSORPTION; AUTOLYSIS; CAPACITY; DIGESTION; ENZYMES; IMMOBILIZED ENZYMES; INACTIVATION; LEACHING; LIPASES; SILICA; STABILITY; STABILIZATION; enzyme stabilization; mesoporous silica; nanoscale enzyme reactors; nanobiotechnology; Environmental Molecular Sciences Laboratory

Citation Formats

Kim, Moon Il, Kim, Jungbae, Lee, Jinwoo, Jia, Hongfei, Na, Hyon Bin, Youn, Jongkyu, Kwak, Ja Hun, Dohnalkova, Alice, Grate, Jay W., Wang, Ping, Hyeon, Taeghwan, Park, Hyun-Gyu, and Chang, Ho Nam. Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization. United States: N. p., 2007. Web. doi:10.1002/bit.21107.
Kim, Moon Il, Kim, Jungbae, Lee, Jinwoo, Jia, Hongfei, Na, Hyon Bin, Youn, Jongkyu, Kwak, Ja Hun, Dohnalkova, Alice, Grate, Jay W., Wang, Ping, Hyeon, Taeghwan, Park, Hyun-Gyu, & Chang, Ho Nam. Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization. United States. doi:10.1002/bit.21107.
Kim, Moon Il, Kim, Jungbae, Lee, Jinwoo, Jia, Hongfei, Na, Hyon Bin, Youn, Jongkyu, Kwak, Ja Hun, Dohnalkova, Alice, Grate, Jay W., Wang, Ping, Hyeon, Taeghwan, Park, Hyun-Gyu, and Chang, Ho Nam. Thu . "Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization". United States. doi:10.1002/bit.21107.
@article{osti_898617,
title = {Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization},
author = {Kim, Moon Il and Kim, Jungbae and Lee, Jinwoo and Jia, Hongfei and Na, Hyon Bin and Youn, Jongkyu and Kwak, Ja Hun and Dohnalkova, Alice and Grate, Jay W. and Wang, Ping and Hyeon, Taeghwan and Park, Hyun-Gyu and Chang, Ho Nam},
abstractNote = {alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.},
doi = {10.1002/bit.21107},
journal = {Biotechnology and Bioenegineering, 96(2):210-218},
number = ,
volume = ,
place = {United States},
year = {Thu Feb 01 00:00:00 EST 2007},
month = {Thu Feb 01 00:00:00 EST 2007}
}
  • : Hierarchically ordered mesocellular mesoporous silica materials (MMS) were synthesized using a single structure directing agent under neutral conditions for the first time. The mesocellular pores are synthesized without adding any pore expander, and the walls of cellular pores in MMS are composed of SBA-15 type mesopores. The small-angle X-ray scattering (SAXS) pattern of MMS revealed the presence of ordered pore structures with two different length scales. The current MMS possesses four different pore systems; complementary micro/mesopores, main 13 nm mesopores, 40 nm mesocellular spherical pores, and textural inter-particle macropores. Nanometer-scale enzyme reactors (NER) were developed in mesocellular mesoporous silicamore » (MMS) via a ship-in-a-bottle approach, which employs adsorption of enzymes followed by cross-linking using glutaraldehyde (GA) treatment. The resulting NER show an impressive stability and activity with an extremely high loading of enzymes. For example, NER containing α-chymotrypsin (NER-CT) could hold 0.5 g CT in 1 g of silica, but the specific activity of NER-CT was 10.4 times higher than that of the adsorbed CT with a lower loading (0.07 g CT per 1 g of silica), which was further decreased by a continuous leaching of adsorbed CT. NER-CT showed excellent stability without any leaching, i.e. no activity decrease at all in a rigorously-shaking condition for two weeks (a half-life with 3.8 years), while the conventional adsorption method resulted in a half-life of 3.6 days in the same condition.« less
  • Magnetically-separable and highly-stable enzyme system was developed by adsorption of enzymes in superparamagnetic hierarchically ordered mesocellular mesoporous silica (M-HMMS) and subsequent enzyme crosslinking. Superparamagnetic nanoparticles were homogeneously incorporated into hierarchically-ordered mesocellular mesoporous silica (HMMS) by the decomposition of preformed iron propionate complex. The size of incorporated superparamagnetic 15 nanoparticles was around 5 nm, generating a magnetically separable host with high pore volumes and large pores (M-HMMS). α-chymotrypsin (CT) was adsorbed into M-HMMS with high loading (~30 wt%) in less than 30 minutes. Glutaraldehyde (GA) treatment of adsorbed CT resulted in nanometer scale crosslinked enzyme aggregates in M-HMMS (CLEA-M). The activitymore » of these CT aggregates in M-HMMS (CLEA-M-CT) was 34 times than that of simply adsorbed CT in M20 HMMS, due to an effective prevention of enzyme leaching during washing via a ship-in-a-bottle approach. CLEA-M-CT maintained the intial activity not only under shaking (250 rpm) for 30 days, but also under recycled uses of 35 times. The same approach was employed for the synthesis of CLEA-M of lipase (CLEA-M-LP), and proven to be effective in improving the loading, activity, and stability of enzyme when compared to those of adsorbed LP in M-HMMS.« less
  • Enzymes are versatile nanoscale biocatalysts, and find increasing applications in many areas, including organic synthesis[1-3] and bioremediation.[4-5] However, the application of enzymes is often hampered by the short catalytic lifetime of enzymes and by the difficulty in recovery and recycling. To solve these problems, there have been a lot of efforts to develop effective enzyme immobilization techniques. Recent advances in nanotechnology provide more diverse materials and approaches for enzyme immobilization. For example, mesoporous materials offer potential advantages as a host of enzymes due to their well-controlled porosity and large surface area for the immobilization of enzymes.[6,7] On the other hand,more » it has been demonstrated that enzymes attached on magnetic iron oxide nanoparticles can be easily recovered using a magnet and recycled for iterative uses.[8] In this paper, we report the development of magnetically-separable and highly-stable enzyme system by the combined use of two different kinds of nanostructured materials: magnetic nanoparticles and mesoporous silica.« less
  • Epidermal growth factor receptor antibody (EGFRAb) conjugated silica nanorattles (SNs) were synthesized and used to develop receptor mediated endocytosis for targeted drug delivery strategies for cancer therapy. The present study determined that the rate of internalization of silica nanorattles was found to be high in lung cancer cells when compared with the normal lung cells. EGFRAb can specifically bind to EGFR, a receptor that is highly expressed in lung cancer cells, but is expressed at low levels in other normal cells. Furthermore, in vitro studies clearly substantiated that the cPLA{sub 2}α activity, arachidonic acid release and cell proliferation were considerablymore » reduced by pyrrolidine-2 loaded EGFRAb-SN in H460 cells. The cytotoxicity, cell cycle arrest and apoptosis were significantly induced by the treatment of pyrrolidine-2 loaded EGFRAb-SN when compared with free pyrrolidine-2 and pyrrolidine-2 loaded SNs in human non-small cell lung cancer cells. An in vivo toxicity assessment showed that silica nanorattles and EGFRAb-SN-pyrrolidine-2 exhibited low systemic toxicity in healthy Balb/c mice. The EGFRAb-SN-pyrrolidine-2 showed a much better antitumor activity (38%) with enhanced tumor inhibition rate than the pyrrolidine-2 on the non-small cell lung carcinoma subcutaneous model. Thus, the present findings validated the low toxicity and high therapeutic potentials of EGFRAb-SN-pyrrolidine-2, which may provide a convincing evidence of the silica nanorattles as new potential carriers for targeted drug delivery systems. - Highlights: • EGFRAb-SN developed for receptor-mediated Drug delivery system (DDS). • EGFRAb-SN-pyrrolidine-2 targeted DDS for cPLA2α inhibition in NSLC. • Study indicates EGFRAb-SN-pyrrolidine-2 as an efficient in target dug delivery carrier. • Study explains entire efficiency of EGFRAb-SN-pyrrolidine-2 in vitro and in vivo models.« less
  • The following work was acknowledged at EMSL: α-Chymotrypsin (CT) and lipase (LP) were immobilized in SBA-15 mesoporous silica by crosslinking adsorbed enzymes. This simple approach resulted in one-dimensional crosslinked enzyme aggregates (CLEAs) in the linear pore channels of SBA-15, which was very effective in preventing the enzyme leaching and consequently improving the enzyme stability. Both CLEAs of CT and LP showed negligible activity decrease under harsh shaking condition for one week while the conventional approaches including adsorption and covalent attachment resulted in more than 50–90% enzyme inactivation under the same condition. This effective stabilization results from the bent pore structuremore » of SBA-15 with a high aspect ratio, which prevents the leaching of one-dimensional CLEAs and thereby achieves the higher enzyme loading capacity. Along with the higher specific activity than that of adsorbed enzymes, this CLEA approach is much simpler than that of covalent attachment by obviating the tedious processes for silica functionalization and enzyme attachment.« less