Thermostabilization of Candida antarctica lipase B by double immobilization: Adsorption on a macroporous polyacrylate carrier and R1 silaffin-mediated biosilicification

Process Biochemistry 48 (2013) 1181–1187

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Thermostabilization of Candida antarctica lipase B by double immobilization: Adsorption on a macroporous polyacrylate carrier and R1 silaffin-mediated biosilicification Chanha Jun a,1 , Byoung Wook Jeon a,1 , Jeong Chan Joo a , Quang Anh Tuan Le a , Sol-A. Gu a , Sungmin Byun a , Dae Haeng Cho a , Dukki Kim b , Byoung-In Sang c,∗ , Yong Hwan Kim a,∗∗ a

Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea Value Creation Center, GS Caltex Corporation, Munji-Dong, Yuseong-Gu, Deajeon 305-308, Republic of Korea c Department of Chemical Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 133-791, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 21 August 2012 Received in revised form 17 May 2013 Accepted 7 June 2013 Available online 15 June 2013 Keywords: Candida antarctica lipase B Silaffin R1 peptide Biosilicification Immobilization Thermostability

a b s t r a c t A large improvement in the thermostability of Candida antarctica lipase B (CALB) was achieved through double immobilization, i.e., physical adsorption and R1 silaffin-mediated biosilicification. The C-terminus of CALB was fused with the R1 silaffin peptide for biosilicification. The CALB-R1 fusion protein was adsorbed onto a macroporous polyacrylate carrier and then subsequently biosilicified with tetramethyl orthosilicate (TMOS). After R1 silaffin-mediated biosilicification, the double-immobilized CALB-R1 exhibited remarkable thermostability. The T50 60 of the double-immobilized CALB-R1 increased dramatically from 45 to 72 ◦ C and that was 27, 13.8, 9.8 and 9.9 ◦ C higher than the T50 60 values of free CALB-R1, CALB-R1 adsorbed onto a resin, commercial Novozym 435, and Novozym 435 treated with TMOS, respectively. In addition, the time required for the residual activity to be reduced to half (t1/2 ) of the double immobilized CALB-R1 elevated from 12.2 to 385 min, which is over 30 times longer life time compared free CALB-R1. The optimum pH for biosilicification was determined to be 5.0, and the double-immobilized enzyme showed much better reusability than the physically adsorbed enzyme even after 6 repeated reuses. This R1-mediated biosilicification approach for CALB thermostabilization is a good basis for the thermostabilization of industrial enzymes that are only minimally stabilized by protein engineering. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Candida antarctica lipase B (CALB) has drawn much attention due to its high stability, stereoselectivity, and enantiopreference properties that make it superior to other lipases in biotransformation (E.C. 3.1.1.3). CALB is thus used in a wide variety of industrial fields and is also commercially available (e.g., Novozym 435) [1,2]. However, despite the intrinsic stability of CALB, further thermostabilization is necessary to improve the efficiency of biocatalytic processes at high temperatures. For example, the CALB-catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate exhibits a low efficiency and a reduced reaction rate at temperatures over 60 ◦ C [3]. Lipase-catalyzed biodiesel production is not efficient at a low temperature because many types of

∗ Corresponding author. ∗∗ Corresponding author. Tel.: +82 2 940 5675; fax: +82 2 941 1785. E-mail addresses: [email protected] (B.-I. Sang), [email protected] (Y.H. Kim). 1 These authors contributed equally to this work. 1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2013.06.010

triacylglycerols are highly viscous at low temperatures [4,5]. The cost of the enzyme is a key determinant of the economic feasibility of biotransformations. Therefore, highly thermostable CALB is desirable in reducing the cost of biocatalysts and improving the economic feasibility of biotransformation processes. Protein engineering has been the most popular and powerful tool used to improve the thermostability of proteins [6–11]. In the case of CALB, the catalytic properties can be engineered successfully [12,13]; however, the improvement in thermostability is marginal, and no mutants have exhibited thermal resistance suitable for use at high temperatures (>60 ◦ C) [14–16]. Our group created a thermostable CALB in silico, but the protein-engineered CALB showed a low residual activity of only 20% at temperatures exceeding 60 ◦ C [14]. The difficulty in engineering the thermostability of CALB necessitates the development of a thermostabilization approach other than protein engineering. Enzyme immobilization is a conventional but effective method to improve thermostability [17]. In particular, sol–gel entrapment is used widely due to its mild reaction conditions. Various methods for sol–gel formation have been proposed for building nanostructures and the immobilization of enzymes [18]. However, most

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reagents used in silica sol–gel formation require acidic or alkaline pH conditions, which may disrupt the structure of proteins during sol–gel formation [19]. Silaffin peptides, which are associated with the silica nanostructures in diatoms found in nature, can catalyze the hydrolysis of TMOS to form a silica matrix at room temperature and encapsulate enzymes in a silica matrix [20,21]. Many silaffin peptides (R1–R7) are available for the nano-scaled biosilicification of proteins. According to our previous study, the R1 peptide (SSKKSGSYYSYGTKKSGSYSGYSTKKSASRRIL) is the most powerful fusion partner for the biosilicification of proteins [22]. In this study, CALB was fused with R1 silaffin (CALB-R1) and immobilized through two steps: the CALB-R1 fusion protein was first immobilized on macroporous polyacrylate carriers through physical adsorption and was then biosilicified using TMOS as a precursor. The resulting CALB-R1 (hereafter, “double-immobilized CALB-R1”) was tested to measure its thermal tolerance. 2. Materials and methods

The loading content of CALB-R1 on the supporting material was estimated to be 40 ␮g-(CALB-R1)/mg-(supporting carrier). 2.4. Biosilicification of the immobilized CALB-R1 and Novozym 435 by using TMOS CALB-R1 immobilized on Lewatit VP OC 1600 resin and commercial Novozym 435 were mixed with 2 mL of citrate-(disodium phosphate) buffer at various pHs at room temperature respectively. TMOS was pre-hydrolyzed with 1 mM HCl solution and added to the immobilized CALB-R1 solution and Novozym 435 solution at a final concentration of 100 mM for 15 min at room temperature [25]. The doubleimmobilized CALB-R1 and Novozym 435 were separated by filtration and washed twice with a buffer of each pH value. The wet immobilized CALB-R1 and Novozym 435 were dried in a desiccator at 4 ◦ C for 24 h. 2.5. Analysis of silica deposition on the supporting carrier using SEM–EDX with FIB (focused ion beam) A Quanta 3D focused ion beam/field emission gun with an energy-dispersive Xray spectroscopy (EDX) detector (FEI Company, USA) was used to analyze the silica deposited on the supporting carrier. For image scanning, an accelerating voltage of 5 kV was used, but a signal was obtained using a voltage of 15 kV during the EDX analysis.

2.1. Materials 2.6. Enzyme assay

Pichia pastoris X-33 and the pPICZ␣A plasmid were purchased from Invitrogen (USA). The CALB gene (NCBI gi: 515791) was synthesized by GenScript (USA). The QIAquickTM PCR purification kit was obtained from Qiagen (USA). DH5␣ competent cells were purchased from RBC Bioscience (Taiwan). Bradford reagent for enzyme quantification was purchased from Sigma–Aldrich (USA). All reagents containing para-nitrophenyl butyrate (pNPB) and tetramethyl orthosilicate (TMOS) were purchased from Sigma–Aldrich (USA). PCR primers were synthesized by Cosmo Genetech (South Korea). Macroporous polyacrylate (Lewatit VP OC 1600) was purchased from Bayer (Germany).

Enzyme activity was measured in 50 mM Tris–HCl buffer (pH 8.0) containing 0.1 mM pNPB [26,27]. Enzyme (free CALB-R1: 5–10 ␮g, CALB-R1 immobilized on VP OC: 5 mg, Novozym 435: 5 mg or double-immobilized CALB-R1: 5 mg) was added to 15 mL of a 0.1 mM pNPB solution in 50 mM Tris–HCl buffer (pH 8.0) and incubated for 30 min at 30 ◦ C. The resulting para-nitrophenol was detected by UV/VIS spectroscopy at 405 nm. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 ␮mol of pNPB per minute under standard conditions (30 ◦ C and pH 8.0).

2.2. Expression and purification of CALB-R1

2.7. Measurement of the thermostability of free and immobilized CALB-R1

The C-terminus of CALB was fused with the R1 silaffin domain, and a His6 -tag was added to the N-terminus to allow for purification with a Ni-NTA column (NiNTA Fast Start Kit, Qiagen). The expression vector was transformed into P. pastoris X-33 cells by electroporation [23]. The transformants were cultivated as a seed culture in 50 mL of buffered minimal glycerol-complex medium (BMGY: 10 g of yeast, 20 g of peptone, 700 mL of 1 M potassium phosphate buffer at pH 6.0, 2 mL of 13.4% yeast nitrogen base (YNB), 100 mL of 10% glycerol, and 1 L of distilled water) and inoculated into basal salt medium (BSM: 54 mL of 85% H3 PO4 , 1.86 g of CaSO4 , 36.4 g of K2 SO4 , 29.8 g of MgSO4 ·7H2 O, 8.26 g of KOH, 80 g of glycerol, and 2 L of distilled water). A BioFlo 310 fermenter (New Brunswick Scientific, USA) was used for the expression of CALB-R1 for 96 h at 28 ◦ C and pH 5.0, and protein expression was induced by adding 100% methanol to a final concentration of 0.5% methanol every 24 h to maintain induction according to the manufacturer’s instructions (EasySelect pichia Expression Kit, Invitrogen). The level of dissolved oxygen was maintained at 30% saturation or above by supplying air and pure oxygen and agitating the system at a speed of 500–1100 rpm to meet the oxygen demand. For dry cell mass determination, 10 mL of culture samples were collected, filtrated on 0.45 ␮m filter, washed with distilled water and dried at 95 ◦ C for 24 h until little weight difference was detected. The supernatant containing the target enzyme was harvested by removing the cells by centrifugation at 4500 × g for 10 min at 4 ◦ C. The CALB-R1 in the supernatant was purified as described previously [24]. Affinity chromatography (Ni-NTA agarose, Qiagen, USA) was used to purify the CALB-R1 in the supernatant. The pH of the supernatant was adjusted to pH 8.0 using 1 M NaOH. The formed precipitate was then removed by centrifugation at 4500 × g for 10 min at 4 ◦ C. The supernatant containing CALB-R1 was incubated with a Ni-NTA agarose bead at 4 ◦ C for 1 h and loaded onto the column. The Ni-NTA agarose bead was washed with a washing buffer (50 mM NaH2 PO4 , 20 mM imidazole, 300 mM NaCl, pH 8.0), and CALB-R1 was eluted with an elution buffer (50 mM NaH2 PO4 , 250 mM imidazole, 300 mM NaCl, pH 8.0). The protein concentrations were determined using Bradford reagent according to the manufacturer’s instructions.

In order to elucidate the thermal inactivation and thermostability of free and immobilized CALB-R1, 5–10 ␮g free CALB-R1, 5 mg CALB-R1 immobilized on VP OC, 5 mg Novozym 435, and 5 mg double-immobilized CALB-R1 was added to PCR tube containing 100 ␮L of 50 mM Tris–HCl buffer (pH 8.0), respectively. The thermal inactivation of free and immobilized CALB-R1 was assessed by incubation at 60 ◦ C for different durations ranging from 0 to 60 min, with subsequent cooling in ice for 10 min. The thermal inactivation constants of free and immobilized CALB-R1 were determined by estimating the thermal inactivation rate constant (kd ) and half-life (t1/2 ). All data analyses were performed by linear regression fitting. The thermostability of free and immobilized CALB-R1 was determined by measuring the residual activity after incubation at different temperatures. Each sample in 50 mM Tris–HCl buffer (pH 8.0) was incubated at temperatures of 30–90 ◦ C for 1 h and then cooled for 10 min on ice, after which the enzyme activity was determined. The T50 60 is defined as the temperature at which half of the enzyme activity remains after 1 h of incubation relative to the activity remaining after 1 h of incubation at 30 ◦ C. All experimental results were the average values of triplicate measurements.

2.3. Adsorption of CALB-R1 on a macroporous polyacrylate carrier

CALB fused with R1 was expressed successfully in the Pichia pastoris X-33 system. The lipase activity measured using pNPB started to increase after 48 h of cultivation and approached 2.5 U/mL (Fig. 1). The expression of recombinant CALB-R1 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (Fig. 2). The gel showed a single band corresponding to CALB-R1 (approximately 37 kDa). The specific activity of purified CALB-R1 was similar (20 U/mg) to that previously reported (30 U/mg) [28].

After fed-batch fermentation for the expression of CALB-R1, the supernatant solution was buffer exchanged with 50 mM Tris–HCl buffer at pH 8.0 using an Amicon Ultra Filter with a molecular-weight cutoff of 10,000 (AMICON® Ultra, Millipore). The concentration of CALB-R1 was quantified using the Bio-Rad Protein Assay kit, with bovine serum albumin as the standard. Lewatit VP OC 1600 (Bayer) was used as the supporting material for the immobilization of CALB-R1. Fifty milligrams of CALB-R1 was immobilized on 1.25 g of Lewatit VP OC 1600. Before immobilization through physical adsorption, the Lewatit VP OC 1600 was pre-wetted with ethanol and then mixed with buffer-exchanged CALB-R1 and incubated for 48 h at 25 ◦ C.

2.8. Reusability of immobilized CALB-R1 The reusability of immobilized enzyme was measured by repeated use in a reaction mixture. After each reaction, the immobilized enzyme was recovered from the reaction mixture by filtration with Whatman paper (pore size 0.2 ␮m) and washed with 10 ml of 50 mM Tris–HCl buffer (pH 8.0). Then, the immobilized enzyme was used for another reaction cycle using a fresh reaction mixture.

3. Results and discussion 3.1. Expression and purification of CALB-R1

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Table 1 Thermal inactivation parameters at 60 ◦ C. Enzymes

kd [min−1 ]

t1/2 a [min]

Free CALB-R1 CALB-R1 immobilized on VP OC Double-immobilized CALB-R1 Novozym 435 Novozym 435 treated with TMOS

0.057 0.014 0.002 0.015 0.012

12.2 50.9 385 46.5 58.3

a

Fig. 1. Effect of the incubation time on the cell density and enzyme activity of Pichia pastoris X-33 containing CALB-R1: (a) dry cell weight (), optical density at 600 nm (), and lipase activity ().

3.2. Double immobilization and analysis of silica deposition on the supporting carrier The protein quantification and analysis of the residual activity of CALB-R1 in the aqueous immobilization medium revealed that 85% of free CALB-R1 (42.5 mg) was adsorbed on the Lewatit VP OC 1600 resin. The immobilized CALB-R1 was biosilicified with 100 mM TMOS. The level of silica deposition on the supporting carrier was investigated using SEM–EDX. As shown in Fig. 3(a) and (b), no silicon atoms were observed on the surface of the supporting carrier. A FIB was used to remove the surface layer of the carrier. Fig. 3(c) and (d) clearly shows that the synthesized silica was located only inside the supporting carrier. The supporting carrier (Lewatit VP OC 1600) used in this study is highly macroporous [29]. Most of the CALB-R1 can be adsorbed on the surface of the macropores because their diameter (>100 nm) is sufficiently large for CALB-R1 to pass

Fig. 2. Expression and purification of CALB-R1. SDS-PAGE analysis: lane M: molecular mass markers; (1) culture supernatant; (2) purified CALB-R1.

t1/2 = ln 2/kd (kd : first-order rate constant of inactivation).

through (Scheme 1). The adsorbed CALB-R1 on the macroporous surface may catalyze the formation of a silica matrix from TMOS on the surface, as observed by SEM–EDX after the removal of the surface layer. Enzyme immobilization through conventional sol–gel processes can result in the formation of excessive silica matrix on enzymes, which may cause serious mass transfer resistance [30]. In contrast, biosilicification may have a less severe mass transfer problem because the enzymes are coated with a very thin layer of the silica matrix, as shown in Fig. 3. It was reported that the coupling efficiency (effectiveness factor) of biosilicification-based encapsulation of CALB-R5 was 14% [31]. However, the coupling efficiency of double immobilized CALBR1 was determined as 2.3%, which seems lower compared with reported one (14%). The low value of our double-immobilized CALBR1 could be resulted from the difference of silaffin peptide (R1 vs R5) or immobilization method (direct encapsulation in silica vs double immobilization). Even though we tried to obtain the biosilicificated insoluble with free CALB-R1, the sufficient amount of insoluble particles was not achieved. According to the previous report, a very fine and nano scale beads are formed during the biosilicification of GFP-R1, which could cause difficult to separate and prepare the large amount of insoluble particles [22]. 3.3. Influence of biosilicification on the thermostability of CALB-R1 The thermal inactivation rate constants (kd ) of free and immobilized CALB-R1 were determined from the slope of the logarithmic plot of activity against time (Fig. 4). The time required for the residual activity to be reduced to half (t1/2 ) of that of free, physically immobilized CALB-R1, double-immobilized CALB-R1, Novozym 435 and Novozym 435 treated with pre-polymerized TMOS at 60 ◦ C was calculated to be 12.2, 50.9, 385, 46.5 and 58.3 min, respectively (Table 1). This means that the double-immobilized CALB-R1 required a much longer life time to be thermally inactivated at 60 ◦ C compared with the other enzymes. The adsorption of CALB-R1 on the Lewatit VP OC 1600 improved the enzyme’s thermostability, but double immobilization resulted in a greater improvement in the thermostability of CALB-R1 (Fig. 5). Free CALB-R1 maintained a residual activity of less than 40% after incubation for 1 h at 50 ◦ C, and the T50 60 value was approximately 45 ◦ C. Free CALB-R1 had 18% residual activity after incubation at 70 ◦ C. CALB-R1 adsorbed onto the supporting carrier had higher thermostability than free CALB. The T50 60 value of adsorbed CALBR1 was approximately 58 ◦ C, and 32% of the activity was retained after incubation at 70 ◦ C. Many commercially available lipases are immobilized on supporting carriers to improve their stability. The thermostability of Novozym 435 was also measured to analyze the thermostabilization effect of our method. The T50 60 value of Novozym 435 was slightly higher compared CALB-R1 adsorbed onto the supporting carrier, which may imply that commercial CALB of Novozym 435 has slightly higher thermostability rather that of CALB-R1. However, it was not possible to compare directly the thermostability of two enzymes since it is not easy to obtain free CALB from Novozym 435. Novozym 435 and TMOS treated

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Fig. 3. Analysis of silica deposition in the supporting carrier using SEM–EDX with FIB: (a) SEM image of the supporting carrier surface after biosilicification, (b) elemental composition of the surface of the supporting carrier, (c) SEM image of a cross section of the supporting carrier after biosilicification, and (d) elemental composition of a cross section of the supporting carrier.

Fig. 4. Thermal inactivation of free and immobilized CALB-R1 at 60 ◦ C. Residual activity was measured at 30 ◦ C, pH 8.0. Freely dissolved CALB-R1 (䊉), CALB-R1 immobilized on VP OC (), double-immobilized CALB-R1 at pH 5.0 (), Novozym 435 () and Novozym 435 treated with TMOS ().

Fig. 5. Influence of immobilization on thermostability: freely dissolved CALB-R1 (䊉), CALB-R1 immobilized on VP OC (), double-immobilized CALB-R1 at pH 5.0 (), Novozym 435 () and Novozym 435 treated with TMOS ().

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Scheme 1. The immobilization of CALB-R1 using physical adsorption and biosilicification.

Novozym 435 showed very similar values of T50 60 around 62 ◦ C and each enzyme foliation exhibited 24% and 29% residual activity after incubation at 70 ◦ C. It implies that Novozym 435 was not biosilicified with 100 mM TMOS appropriately at our experimental condition. That may be reason why Novozym 435 treated with pre-polymerized TMOS did not show the drastic improvement of thermostability. However, compared with the single immobilization of CALB-R1, the biosilicification of immobilized CALB-R1 on a carrier surface dramatically improved the thermostability of CALBR1. The double-immobilized CALB-R1 had a T50 60 value of 72 ◦ C and exhibited 58% residual activity after incubation at 70 ◦ C. In general, a 5–8 ◦ C increase in the T50 60 value through protein engineering is a respectable achievement [32], but the protein engineering of CALB failed to reach this standard. However, our group recently reported that thermostable CALB engineered through rational disulfide bond design had a T50 60 value 8.5 ◦ C higher than the value for the wild-type enzyme [33]. In the present study, the double-immobilized CALB-R1 exhibited a T50 60 value 17 ◦ C higher than that of the disulfide bond-designed CALB. These results prove that double immobilization by adsorption and biosilicification is much more effective than other immobilization or protein engineering approaches for the thermostabilization of CALB. Enzyme immobilization can limit deleterious motion at high temperature, which might reduce the unfolding rate or the probability of aggregate formation, thus leading to improved thermostability [17,34]. In our double immobilization approach, the physical adsorption could restrict the motion of local structures, and biosilicification could then further stabilize the immobilized CALB-R1 by encapsulating the overall structure of the fusion protein with silica (Scheme 1). The single immobilization of enzymes by adsorption or sol–gel entrapment often has the drawback of enzyme leakage from the supporting carrier [35,36]. For example,

Table 2 Si content of a cross section of the supporting carrier according to the pH used for biosilicification. pH for biosilicification

Si content [Si/C, %]a

T50 60 [◦ C]

pH 5.0 pH 6.0 pH 7.0

12.46 9.90 5.58

72 50 46

a The Si content was determined SEM–EDX after removal of the supporting carrier using a FIB.

Novozym 435 suffers from the physical desorption of CALB during reactions [37]. Therefore, biosilicified encapsulation could be a good approach to provide a synergistic thermostabilization effect on single-immobilized enzymes by preventing enzymes from undergoing physical desorption. 3.4. Influence of pH on biosilicification Although CALB exhibited optimum activity at pH 8.0, the silaffincatalyzed synthesis of the silica matrix was influenced by the pH because the relationship between the pI of the target enzyme and the pH of the environment affects the biosilicification efficiency [38]. The T50 60 values of the double-immobilized CALB-R1 depended on the pH used for biosilicification (Table 2). The biosilicification of CALB-R1 adsorbed on the resin at pH 5.0 was the most effective for improving the thermostability of CALB (T50 60 ≈ 72 ◦ C). The T50 60 values of the biosilicified CALB-R1 were approximately 50 and 46 ◦ C at pHs 6.0 and 7.0, respectively. The scanning electron microscopy–electron dispersive X-ray spectroscopy (SEM–EDX) data also support the relationship between the thermostability of CALB-R1 and silica deposition, as shown in Table 2. After biosilicification at pH 5.0, the supporting carrier was coated with the highest

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(2011K000660), the National Research Foundation of Korea (NRF20110029249) and Kwangwoon University 2013.

References

Fig. 6. The usability of the immobilized CALBs: CALB-R1 immobilized on VP OC (䊉), double-immobilized CALB-R1 at pH 5.0 (), and Novozym 435 ().

concentration of deposited silica matrix around CALB-R1, which could contribute to the prevention of enzyme leakage from the supporting carrier during reactions. This result demonstrates that the synthesis of silica mediated by the R1 silaffin peptide was influenced by the pH and that a large amount of synthesized silica matrix around CALB-R1 could result in the further thermostabilization of CALB-R1. 3.5. The reusability of the immobilized CALB-R1 The repeated use of immobilized enzymes is the most important requirement for industrial applications, and the reusability of immobilized CALB was investigated in this study (Fig. 6). Novozym 435 and CALB-R1 adsorbed on the resin showed almost the same reusability. The physically immobilized CALB enzymes exhibited less than 30% of their initial activity after five cycles. The hydrolysis reaction of pNPB at 30 ◦ C was not deleterious to the thermostability of the enzymes, and thus, the decreased activity of the physically immobilized CALB enzymes can be attributed to the leaching of the enzymes during the washing step. In contrast to the physically immobilized CALB enzymes, the double-immobilized CALB-R1 retained almost 100% of its initial activity after five cycles, indicating that biosilicification could physically prevent the adsorbed enzymes from leaking out of the supporting carrier. 4. Conclusions The thermostability of CALB-R1 was successfully improved using a combined immobilization method, i.e., physical adsorption and biosilicification. The silica matrix could entrap the CALB-R1 adsorbed on the macroporous polyacrylate carrier and effectively prevent the inactivation of CALB-R1 at high temperatures, resulting in an improvement of 27 ◦ C in the Tm value. The SEM–EDX analysis revealed that the synthesized silica matrix was distributed around CALB-R1 on the supporting carrier and indicated that the extent of silica deposition was proportional to the thermostability of CALBR1. The resulting CALB-R1 with enhanced thermostability would be useful for a wide range of applications, such as biodiesel production and other lipase-catalyzed synthesis processes. Acknowledgments This research was supported by the R&D program of MKE/KEIT (10031717), the Converging Research Center Program

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