Preparation and characterization of polymer-coated mesoporous silica nanoparticles and their application in Subtilisin immobilization

Belma Özbek,Şule Ünal 한국화학공학회 2017 Korean Journal of Chemical Engineering Vol.34 No.7

The preparation and characterization of polymer-coated mesoporous silica nanoparticles (MSNs) and their application in Subtilisin (Alcalase®) immobilization were investigated. For the synthesis of polymer-coated MSNs, acrylic acid (AA) and chitosan (CS) mixture were blended as poly(acrylic acid) (PAA) and CS polymer layer onto MSNs via in-situ polymerization technique. Then, both uncoated MSNs and polymer-coated mesoporous silica nanoparticles (CS-PAA/MSNs) were characterized by taking into account properties such as morphologic pattern, size distribution, surface charge of the particles as well as thermogravimetric stability with SEM, TEM, Zetasizer and TGA analyses. Subtilisin was immobilized onto polymer-coated mesoporous silica nanoparticles via adsorption technique. For optimizing the enzyme immobilization process, the percent enzyme loading depending on the matrix amount, immobilization time and pH were investigated. Then, the activity values of immobilized enzyme and free enzyme were compared at various pH and temperature values. The maximum enzyme activity was achieved at pH 9.0 for both immobilized and free enzyme. Immobilized enzyme showed more stability at higher temperatures compared with free enzyme. Furthermore, the operational and storage stability of immobilized enzyme were determined. The activity of immobilized enzyme was reduced from 100% to 45.83% after five repeated uses. The storage stability of immobilized enzyme was found to be higher than that of free enzyme. The activity of immobilized enzyme was reduced from 100% to 60% after 28 days of storage time. We concluded that the polymer-coated MSNs were a suitable matrix for Subtilisin immobilization compared to uncoated MSNs.

Immobilization of enzymes onto clay minerals for the biochemical decomposition of 4-chlorophenol

( Oh Oh Sung Kwean ),( Su Yeon Cho ),( Jun Won Yang ),( Wooyoun Cho ),( Seonyeong Kwak ),( Sungyoon Park ),( Yejee Lim ),( Han S. Kima ) 한국물환경학회 2017 한국물환경학회·대한상하수도학회 공동 춘계학술발표회 Vol.2017 No.-

In this study an oxidative enzyme was immobilized onto inorganic backbone materials to stimulate the detoxification of toxic aromatic hydrocarbon compounds. Smectite clay minerals and soil organic matter were screened as an enzyme support and a binding agent, respectively. Montmorillonite of which inner pores are layered with nano-scale spacing planar was activated by humic acid. A dioxygenase obtained by cloning of its corresponding gene from Arthrobacter chlorophenolicus A6 was immobilized onto the humic acid-activated montmorillonite. Oxygenated metabolites such as catechol and 4-chlorocatechol were selected as target aromatic contaminants (primary substrates of enzyme). The enzyme immobilization yield was as high as 63% and the reductions in enzyme activity for the decomposition of substrate compounds during enzyme immobilization were minimal: 15% for catechol and 24% for 4-chlorocatechol, respectively. The kinetic analysis of the free and immobilized enzymes demonstrated a slight decrease of vmax and a marginal increase of KM as compared with those for the free enzyme, indicating the changes in enzyme activity perhaps due to the changes in enzyme conformation associated with its immobilization were minimal. The results for the effects of environmental factors including pH, temperature, and ionic strength on the activity of free and immobilized enzymes showed that the activity of free enzyme changed significantly in response to the changes of the environmental factors whereas that of immobilized enzyme was pretty much consistent. This indicated that the stability of enzyme against the abrupt changes in environmental factors can be greatly improved by enzyme immobilization. The results of this study support the feasibility of a new environmental fusion technology based on bio-technology and nano-technology for the development of biochemical treatment processes.

Invited Mini Review : Polymer materials for enzyme immobilization and their application in bioreactors

( Yan Fang ),( Xiao Jun Huang ),( Peng Cheng Chen ),( Zhi Kang Xu ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.2

Enzymatic catalysis has been pursued extensively in a wide range of important chemical processes for their unparalleled selectivity and mild reaction conditions. However, enzymes are usually costly and easy to inactivate in their free forms. Immobilization is the key to optimizing the in-service performance of an enzyme in industrial processes, particularly in the field of non-aqueous phase catalysis. Since the immobilization process for enzymes will inevitably result in some loss of activity, improving the activity retention of the immobilized enzyme is critical. To some extent, the performance of an immobilized enzyme is mainly governed by the supports used for immobilization, thus it is important to fully understand the properties of supporting materials and immobilization processes. In recent years, there has been growing concern in using polymeric materials as supports for their good mechanical and easily adjustable properties. Furthermore, a great many work has been done in order to improve the activity retention and stabilities of immobilized enzymes. Some introduce a spacer arm onto the support surface to improve the enzyme mobility. The support surface is also modified towards biocompatibility to reduce non-biospecific interactions between the enzyme and support. Besides, natural materials can be used directly as supporting materials owning to their inert and biocompatible properties. This review is focused on recent advances in using polymeric materials as hosts for lipase immobilization by two different methods, surface attachment and encapsulation. Polymeric materials of different forms, such as particles, membranes and nanofibers, are discussed in detail. The prospective applications of immobilized enzymes, especially the enzyme-immobilized membrane bioreactors (EMBR) are also discussed. [BMB reports 2011; 44(2): 87-95]

Cellulose nanofibers for magnetically-separable and highly loaded enzyme immobilization

Je, Hwa Heon,Noh, Sora,Hong, Sung-Gil,Ju, Youngjun,Kim, Jungbae,Hwang, Dong Soo Elsevier 2017 CHEMICAL ENGINEERING JOURNAL -LAUSANNE- Vol.323 No.-

<P><B>Abstract</B></P> <P>Cellulose nanofibers (CNFs) are one of attractive supporting materials for enzyme immobilization due to their unique properties such as high surface area, high porosity and surface carboxyl groups for chemical bonding. In this study, CNFs were prepared via TEMPO-mediated oxidation and physical grinding of cellulose, and further used for the immobilization of α-chymotrypsin (CT) enzyme via four different approaches such as covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically-separable EPC (Mag-EPC). EPC approach consists of three steps: covalent enzyme attachment, enzyme precipitation and crosslinking, while EC represents a control without the step of enzyme precipitation. Amine-functionalized magnetic nanoparticles were added during the enzyme precipitation and crosslinking steps to produce magnetically-separable EPC. The activities of CA, EC, EPC and Mag-EPC were 0.067, 0.14, 1.3 and 2.6 units per mg CNFs, respectively, representing that the activity of Mag-EPC was 38-, 19- and 2-times higher than those of CA, EC and EPC, respectively. After incubation under shaking (200rpm) for 30days, CA, EC, EPC and Mag-EPC maintained 12%, 46%, 77% and 50% of their initial activities, respectively, while free CT showed only 0.2% of its initial activity even after 8days. Because CT is a tricky enzyme to stabilize due to its inactivation mechanism via autolysis, the present results of stable EPC and Mag-EPC on CNFs have demonstrated the great potential of CNFs as an environmentally-friendly and economical carrier of enzyme immobilization, which allows for magnetic separation as well as high enzyme activity/loading and stability.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Cellulose nanofibers (CNFs) were used as a carrier of chymotrypsin (CT) immobilization. </LI> <LI> Four different CT immobilization approaches on CNFs were performed. </LI> <LI> CT, a tricky enzyme to stabilize, was stably and massively immobilized on CNFs. </LI> <LI> Magnetic separation of enzyme precipitate coating on CNFs was effective. </LI> <LI> Potential of CNFs for stable and recyclable enzyme immobilization was demonstrated. </LI> </UL> </P>

Single enzyme nanoparticles armored by a thin silicate network: Single enzyme caged nanoparticles

Hong, Sung-Gil,Kim, Byoung Chan,Na, Hyon Bin,Lee, Jinwoo,Youn, Jongkyu,Chung, Seung-Wook,Lee, Chang-Won,Lee, Byoungsoo,Kim, Han Sol,Hsiao, Erik,Kim, Seong H.,Kim, Byung-Gee,Park, Hyun Gyu,Chang, Ho Na Elsevier 2017 Chemical engineering journal Vol.322 No.-

<P><B>Abstract</B></P> <P>For the encapsulation of biomolecules in inorganic materials, we have developed a unique enzyme-silicate conjugate material that consists of a self-assembled molecularly thin silicate layer on the surface of each individual enzyme molecule. The enzyme-silicate conjugate materials, called single enzyme caged nanoparticles (SECNs), were synthesized via the silica polymerization on the surface of enzyme molecule after solubilizing each enzyme molecule in hexane by using a tiny amount of surfactant, called “ion-pairing”. SECNs possess near native enzyme activity in aqueous media with minimal substrate diffusional limitations, and are highly stable under the protection of silicate network cage. Due to their nearly molecular size, SECNs can also be adsorbed into mesoporous silica materials to yield robust and easily-recyclable enzymatic systems that can be used in a number of potential biocatalytic applications such as diagnostics, biosensors, biotransformations, biofuel production, bioremediation and CO<SUB>2</SUB> capture.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Protocol of single enzyme caged nanoparticles (SECNs) has been developed. </LI> <LI> SECNs have ultra-thin silicate network on the surface of individual enzyme molecule. </LI> <LI> SECNs minimize substrate diffusional limitation, and inhibit the enzyme denaturation. </LI> <LI> SECNs can be immobilized into mesoporous silica for recyclable enzymatic systems. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Significant enhancement of direct electric communication across enzyme-electrode interface <i>via</i> nano-patterning of synthetic glucose dehydrogenase on spatially tunable gold nanoparticle (AuNP)-modified electrode

Lee, Hyeryeong,Lee, Yoo Seok,Lee, Soo Kyung,Baek, Seungwoo,Choi, In-Geol,Jang, Jae-Hyung,Chang, In Seop Elsevier 2019 Biosensors & bioelectronics Vol.126 No.-

<P><B>Abstract</B></P> <P>In this study, the effect of inter-enzyme steric hindrance that occurs during enzyme immobilization on the electrode, on direct electrical communications of enzyme with electrode was investigated <I>via</I> nano-patterning of enzymes on the electrode. Here, the nano-patterning of enzymes was achieved through the combination of DET-capable enzyme that was produced <I>via</I> fusion of site-specific gold binding peptide (GBP) to catalytic subunit of enzyme and gold nanoparticle (AuNP) array with highly tunable dimensions of AuNPs, resulting in spatially controllable enzyme-electrode. The nano-scale spatial control between immobilized enzymes on the highly tuned AuNPs shows different DET efficiency across the enzyme-electrode interface, showing 18.47% of maximum electron recovery which is 3.2-fold enhanced electron recovery efficiency compared to spatially non-controlled enzymes on the electrode where showed 5.7% of electron recovery. The result affirms that inter-enzyme interaction is a significant parameter that decides the enzyme-electrode performance.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The fusion of GBP to the catalytic subunit of GDH enabled interfacial DET in the enzyme-electrode. </LI> <LI> The effect of inter-enzyme agglomeration on efficiency of interfacial DET was investigated, related to its effect on <I>R</I> <SUB>ct</SUB>. </LI> <LI> The enzyme nano-patterning was developed via combination of synthetic enzyme and AuNP-modified electrode. </LI> <LI> The nano-scale spatial control of synthetic GDHs on the electrode enhanced electroactive coverage of enzyme-electrode. </LI> <LI> The inter-enzyme agglomeration is the important parameter to consider for the development of DET-based bioelectronics. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Amplicilin biosynthesis by immobilized enzyme

Kim, Young-Sik,Ryu, Dewy-D.Y. The Pharmaceutical Society of Korea 1980 Archives of Pharmacal Research Vol.3 No.1

Ampliciline was synthesized from 6-amino-pencillanic acid (6-APA) and D-.alpha. phenylglycine methyl ester by using amplicilin synthesizing enzyme from Peudomonas melanogenum (IAM 1655). The whole cell enzyme was immobilized by entrapping it in the polyacrylamide gel lattices. The polymer used in the enzyme entrapment was made from 150 mg per ml of acrylamide monomer and 8 mg per ml of N, N'-methylenebisacrylamide. About 200 mg/whole cell enzyme was mixed in the polymer for entrapment. The maximal activity retention after immobilization was 56%. The optimal pH values for the whole cell enzyme and the immobilized whole cell enzyme were 6.0 and 5.9, respectively. The optimal temperature for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and heat increased for immobilized whole cell enzyme. Immobilized cell was more stable especially in the acidic condition while both type were found to be very suceptible to thermal inactivation at a temperature above 4.deg.C. The kinetic constants obtained from Lineweaver-Burk plot based on two substate reaction mechanism showed somewhat higher value for immobilized whole cell enzyme as compared to the whole cell enzyme : the Km value for 6-APA were 7.0 mM and 12.5 mM while Km values for phenylglycine methyl ester were 4.5 mM and 8.2 mM, respectively. Using the immobilized whole cell enzyme packed in a column reactor, the productivity of ampiciline was studied by varying the flow rate of substrate solution. At the space velocity, SV, 0.14 hr$^{-1}$ the conversion was 45%. Operational stability found in terms of half life was 30 hr at SV = 0.2 hr.

Stabilization of Bovine carbonic anhydrase II through rational site-specific immobilization

Lee, Chang Hyun,Jang, Eui Kyoung,Yeon, Young Joo,Pack, Seung Pil Elsevier 2018 Biochemical engineering journal Vol.138 No.-

<P><B>Abstract</B></P> <P>Carbonic anhydrase (CA) is a prominent biocatalyst used for the enzymatic CO<SUB>2</SUB> capture process. For industrial applications, the immobilization of enzyme on a matrix support is a useful technique for stabilizing the enzyme and improving its reusability. Although there have been several trials for immobilization of CA, there is no systematic approach to investigate which site-specific immobilization is more effective for stabilization of CA. In this study, we investigated the effect of the immobilization position on the stability of CA using α-type Bovine CA (bCAII). Six candidate residues (K9, K36, T85, D151, E233, or N252) were selected and each Cys mutant was site-specifically immobilized on the magnetic beads. The thermal and long-term stabilities were compared. Interestingly, the immobilized K9C and K36C, in which the immobilized sites are located close to the N-terminus of bCAII, showed 4.0- and 9.8-fold enhanced thermostability, respectively, at 58 °C. They also maintained 60.6% and 55.5% of activity, respectively, at 45 °C after 20 days, when the wild-type bCAII (free and randomly immobilized) completely lost its activity. These results indicated that the site-specific immobilization of the flexible residues on the N-terminal region could be an effective strategy for the stabilization of bCAII, which would be useful guidelines for the immobilization of other CAs in a site-specific manner.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The effect of the immobilization position on bCAII stability was investigated newly. </LI> <LI> Six mutants, K9C, K36C, T85C, D151C, E233C, and N252C were designed for the site-specific immobilization. </LI> <LI> The immobilized K9C and K36C showed high thermostability and long-term stability. </LI> <LI> Flexible residues near N-terminal region was more proper targets for site-specific immobilization of bCAII. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

충진층 반응기에서 고정화 효소에 의한 난황 단백질의 가수분해

Byung Chul Kang(강병철) 한국생명과학회 2010 생명과학회지 Vol.20 No.11

난황단백질 가수분해를 위한 알칼리성 단백질분해효소를 5가지 담체 Duolite A568, Celite R640, Dowex-1, Dowex 50W 그리고 Silica gel R60 에 고정화하였다. Duolite A568의 경우에 24.7%의 최대 고정화 효율을 나타내었다. 자유 효소와 고정화 효소에 대한 최적의 pH는 각각 8과 9였고, 최적의 pH는 고정화에 의해 염기성으로 1만큼 증가하였다. 그러나 최적 온도는 자유 효소와 고정화 효소 모두 50℃로 같았다. 고정화 효소가 자유 효소에 비해 높은 열 안정성을 보였다. 재사용 회분식 공정에서 10 cycle 동안 효소활성은 초기 활성의 86%를 유지하였다. 연속 공정을 위한 충진층 반응기에서 여러 유속에 대한 장기 조업에서 효소 활성의 안정성 평가하였는데 낮은 유속일수록 높은 활성을 유지하였다. 연속 조업에서 casein과 난황 단백질을 사용하여 원료에 대한 고정화 효소의 활성에 대한 영향을 조사하였다. 96시간 연속 조업에서 casein의 경우는 초기 활성의 83%를 유지하였고 난황 단백질의 경우는 초기 활성의 61%를 유지하였다. Alkaline protease for the hydrolysis of egg yolk protein was immobilized on five carriers ? Duolite A568, Celite R640, Dowex-1, Dowex 50W and Silica gel R60. Duolite A568 showed a maximum immobilization yield of 24.7%. Optimum pH for the free and immobilized enzyme was pH 8 and 9, respectively. However, no change was observed in optimum temperature (50℃). Thermal stability was observed in immobilized enzymes compared to free enzymes. The immobilized enzyme retained 86% activity after 10 cycle operations in a repeated batch process. The effect of flow rate on the stability of enzyme activity in continuous packed-bed reactor was investigated. Lowering flow rate increased the stability of the immobilized enzyme. After 96 hr of continuous operation in a packed-bed reactor, the immobilized enzyme retained 83 and 61% activity when casein and egg yolk were used as a raw materials, respectively.

Immobilization of Keratinolytic Metalloprotease from Chryseobacterium sp. Strain kr6 on Glutaraldehyde-Activated Chitosan

( Silveira Silvana T. ),( Sabrine Gemelli ),( Jeferson Segalin ),( Adriano Brandelli ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.6

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65oC. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.