Jin, Lihua
(College of Bioengineering, Beijing Polytechnic)
,
Li, Ye
(College of Bioengineering, Beijing Polytechnic)
,
Ren, Xiang-Hao
(Key Laboratory of Urban Stormwater System and Water Environment, Ministry of Education, School of Environment and Energy Engineering, Beijing University of Civil Engineering and Architecture)
,
Lee, Jung-Heon
(Department of Chemical and Biochemical Engineering, Chosun University)
Five different polymer nanofibers, namely, polyaniline nanofiber (PANI), magnetically separable polyaniline nanofiber (PAMP), magnetically separable DEAE cellulose fiber (DEAE), magnetically separable CM cellulose fiber (CM), and polystyrene nanofiber (PSNF), have been used for the immobilization of...
Five different polymer nanofibers, namely, polyaniline nanofiber (PANI), magnetically separable polyaniline nanofiber (PAMP), magnetically separable DEAE cellulose fiber (DEAE), magnetically separable CM cellulose fiber (CM), and polystyrene nanofiber (PSNF), have been used for the immobilization of lactase (E.C. 3.2.1.23). Except for CM and PSNF, three polymers showed great properties. The catalytic activities (kcat) of the free, PANI, PAMP, and magnetic DEAE-cellulose were determined to be 4.0, 2.05, 0.59, and 0.042 mM/min·mg protein, respectively. The lactase immobilized on DEAE, PANI, and PAMP showed improved stability and recyclability. PANI- and PAMP-lactase showed only a 0-3% decrease in activity after 3 months of vigorous shaking conditions (200 rpm) and at room temperature (25℃). PANI-, PAMP-, and DEAE-lactase showed a high percentage of conversion (100%, 47%, and 12%) after a 1 h lactose hydrolysis reaction. The residual activities of PANI-, PAMP-, and DEAE-lactase after 10 times of recycling were 98%, 96%, and 97%, respectively.
Five different polymer nanofibers, namely, polyaniline nanofiber (PANI), magnetically separable polyaniline nanofiber (PAMP), magnetically separable DEAE cellulose fiber (DEAE), magnetically separable CM cellulose fiber (CM), and polystyrene nanofiber (PSNF), have been used for the immobilization of lactase (E.C. 3.2.1.23). Except for CM and PSNF, three polymers showed great properties. The catalytic activities (kcat) of the free, PANI, PAMP, and magnetic DEAE-cellulose were determined to be 4.0, 2.05, 0.59, and 0.042 mM/min·mg protein, respectively. The lactase immobilized on DEAE, PANI, and PAMP showed improved stability and recyclability. PANI- and PAMP-lactase showed only a 0-3% decrease in activity after 3 months of vigorous shaking conditions (200 rpm) and at room temperature (25℃). PANI-, PAMP-, and DEAE-lactase showed a high percentage of conversion (100%, 47%, and 12%) after a 1 h lactose hydrolysis reaction. The residual activities of PANI-, PAMP-, and DEAE-lactase after 10 times of recycling were 98%, 96%, and 97%, respectively.
This study examined the stability of lactase immobilized on the following five different polymer nanofibers: polyaniline nanofiber (PANI), polyaniline magnetically separable nanofiber (PAMP), magnetically separable DEAE cellulose fiber (DEAE), magnetically separable CM cellulose fiber (CM), and polystyrene nanofiber (PS). The activities of the free and immobilized lactases were compared under various conditions, including pH and temperature.
대상 데이터
Lactase (E.C.3.2.1.23) from Agaricus bisporus BioChemika powder was purchased from Fluka AG (Switzerland). O-Nitrophenyl-D-galactopyranoside (ONGP), bovine serum albumin standard (BSA), polyaniline, DEAE-cellulose, CM-cellulose, and lactose were obtained from Sigma-Aldrich (St.
이론/모형
The rate of enzymatic hydrolysis of ONPG was expressed using the Michaelis-Menten equation. The kinetic constants (Vmax and Km) were calculated using linear regression analysis based on the least-square method.
The kinetics of the immobilized enzymes was determined using the standard experiments to measure the kinetic parameters using ONPG as the substrate. The rate of enzymatic hydrolysis of ONPG was expressed using the Michaelis-Menten equation.
성능/효과
The thermostability increased with decreasing temperature. After 12 days incubation at 40℃, the retention activity of the lactase immobilized on PANI, PAMP, and DEAE was 96%, 77%, and 57%, respectively.
The results are shown in Table 1. Among the five immobilized lactases, lactase immobilized on PANI and PAMP showed great activity and stability. As shown in Figs.
6% of activity remained after 96 days of exposure to shaking conditions at room temperature. Both PS-lactase and CMC-lactase had a low immobilization yield and low activities. The stability of CM-, and PSlactase was as low as 0 and 17.
A vial reactor with immobilized enzyme was incubated at 37℃ under shaking conditions. For a 1 h reaction, the lactose conversion with PANI-, PAMP-, and DEAE-lactase was 100%, 47%, and 12%, respectively.
In conclusion, the lactases immobilized on PSNF, DEAE, PANI, and PAMP showed improved stability and recyclability. PANI- and PAMP-lactase were quite stable with less than 10% decrease in activity being observed after 3 months of rigorous shaking conditions.
Because of their low activities, the subsequent research did not study PS- and CMClactase. Moreover, DEAE-lactase was better than PS and CMC-lactase, but also showed low activity. In Figs.
4). On the other hand, PSNF- and DEAE-lactase showed low stability with residual activities of 17.6% and 19.8%, respectively, after 2 months under the same conditions. Many studies have reported that immobilized lactase has excellent stability at 4℃ [10].
In conclusion, the lactases immobilized on PSNF, DEAE, PANI, and PAMP showed improved stability and recyclability. PANI- and PAMP-lactase were quite stable with less than 10% decrease in activity being observed after 3 months of rigorous shaking conditions. PANI-, PAMP-, and DEAE-lactase showed a high level of lactose conversion (100%, 47%, and 12%) after a 1 h batch reaction.
PANI- and PAMP-lactase were quite stable with less than 10% decrease in activity being observed after 3 months of rigorous shaking conditions. PANI-, PAMP-, and DEAE-lactase showed a high level of lactose conversion (100%, 47%, and 12%) after a 1 h batch reaction. The immobilized lactases were easily recovered and recycled after the reaction, and their residual activities after recycling 10 times were 98%, 96%, and 97%, respectively.
1A-1F, PANI and PAMP have a large surface area and they can be easily recovered by centrifugation and magnets. The highest immobilization yield (IY, %) obtained with PAMP was 71.8%, and the catalytic activity was 14.5%. The stability of PANI- and PAMP-lactase was quite high, and 95.
PANI-, PAMP-, and DEAE-lactase showed a high level of lactose conversion (100%, 47%, and 12%) after a 1 h batch reaction. The immobilized lactases were easily recovered and recycled after the reaction, and their residual activities after recycling 10 times were 98%, 96%, and 97%, respectively. These immobilized enzymes could be used in lactose analysis and biosensors to detect the lactose concentration.
Table 4 gives a summary of the experimental results published in other papers [4, 9, 10, 15, 23, 27, 31, 34]. The lactases immobilized onto PANI and PAMP showed above 90% residual activity under room temperature and vigorous shaking conditions.
The optimal lactase concentration for immobilization was determined by examining the effects of the lactase concentration in the range of 1-20 mg/ml (20 mM phosphate buffer, pH 6.5) on the immobilization yield and recovery activity. The catalytic activity of the immobilized enzyme was the highest when 5 mg/ml of free lactase was immobilized.
5%. The stability of PANI- and PAMP-lactase was quite high, and 95.1% and 90.6% of activity remained after 96 days of exposure to shaking conditions at room temperature. Both PS-lactase and CMC-lactase had a low immobilization yield and low activities.
후속연구
Since the lactases were immobilized inside the CM pores, there was a problem with mass transfer of the substrate from the bulk solution to the internal pores. Because of their low activities, the subsequent research did not study PS- and CMClactase. Moreover, DEAE-lactase was better than PS and CMC-lactase, but also showed low activity.
참고문헌 (34)
Al-Muftah AE, Abu-Reesh IM. 2005. Effects of internal mass transfer and product inhibition on a simulated immobilized enzyme-catalyzed reactor for lactose hydrolysis. Biochem. Eng. J. 23: 139-153.
Al-Muftah AE, Abu-Reesh IM. 2005. Effects of simultaneous internal and external mass transfer and product inhibition on immobilized enzyme-catalyzed reactor. Biochem. Eng. J. 27: 167-178.
Ansari SA, Husain Q. 2011. Immobilization of Kluyveromyces lactis β-galactosidase on concanavalin A layered aluminium oxide nanoparticles — Its future aspects in biosensor applications. J. Mol. Catal. B Enzym. 70: 119-126.
Bayramoglu G, Tunali Y, Arica MY. 2007. Immobilization of β-galactosidase onto magnetic poly(GMA-MMA) beads for hydrolysis of lactose in bed reactor. Catal. Commun. 8: 1094-1101.
Beyler-Çiğil A, Çakmakçı E, Danış Ö, Demir S, Kahraman MV. 2013. α-Amylase immobilization on modified polyimide material. Chem. Eng. Trans. 32: 1687-1692.
de Lathouder KM, Lozano-Castello D, Linares-Solano A, Kapteijn F, Moulijn JA. 2006. Carbon coated monoliths as support material for a lactase from Aspergillus oryzae: characterization and design of the carbon carriers. Carbon 44: 3053-3063.
de Lathouder KM, Lozano-Castello D, Linares-Solano A, Wallin SA, Kapteijn F, Moulijn JA. 2007. Carbon-ceramic composites for enzyme immobilization. Microporous Mesoporous Mater. 99: 216-223.
Giacomini C, Irazoqui G, Batista-Viera F, Brena BM. 2001. Influence of the immobilization chemistry on the properties of immobilized β-galactosidases. J. Mol. Catal B Enzym. 11: 597-606.
Haider T, Husain Q. 2007. Calcium alginate entrapped preparations of Aspergillus oryzae β-galactosidase: its stability and applications in the hydrolysis of lactose. Int. J. Biol. Macromol. 41: 72-80.
Jimenez-Guzman J, Sarabia-Leos C, Cruz-Guerrero AE, Rodriguez-Serrano GM, Lopez-Munguia A, Gomez-Ruiz L, Garcia-Garibay M. 2006. Interaction between β-lactoglobulin and lactase and its effect on enzymatic activity. Int. Dairy J. 16: 1169-1173.
Jochems P, Mueller T, Satyawali Y, Diels L, Dejonghe W, Hanefeld U. 2015. Active site titration of immobilized β-galactosidase for the determination of active enzymes. Biochem. Eng. J. 93: 137-141.
Numanoglu Y, Sungur S. 2004. β-Galactosidase from Kluyveromyces lactis cell disruption and enzyme immobilization using a cellulose-gelatin carrier system. Process Biochem. 39: 705-711.
Obon JM, Castellar MR, Iborra JL, Manjon A. 2000. [beta]-Galactosidase immobilization for milk lactose hydrolysis: a simple experimental and modelling study of batch and continuous reactors. Biochem. Educ. 28: 164-168.
Ozdural AR, Tanyolac D, Boyaci IH, Mutlu M, Webb C. 2003. Determination of apparent kinetic parameters for competitive product inhibition in packed-bed immobilized enzyme reactors. Biochem. Eng. J. 14: 27-36.
Patel SK, Kalia VC, Choi JH, Haw JR, Kim IW, Lee JK. 2014. Immobilization of laccase on SiO 2 nanocarriers improves its stability and reusability. J. Microbiol. Biotechnol. 24: 639-647.
Peppler HG, Reed G. 1987. Enzymes in food and feed processing. Biotechnology 7a: 578-580.
Pessela BCC, Mateo C, Fuentes M, Vian A, Garcia JL, Carrascosa AV, et al. 2003. The immobilization of a thermophilic β-galactosidase on Sepabeads supports decreases product inhibition: complete hydrolysis of lactose in dairy products. Enzym. Microb. Technol. 33: 199-205.
Sharma SK, Singhal R, Malhotra BD, Sehgal N, Kumar A. 2004. Lactose biosensor based on Langmuir-Blodgett films of poly(3-hexyl thiophene). Biosens. Bioelectron. 20: 651-657.
Song Y-S, Lee H-U, Park C, Kim S-W. 2013. Optimization of lactulose synthesis from whey lactose by immobilized β-galactosidase and glucose isomerase. Carbohydr. Res. 369: 1-5.
Tatavarty R, Hwang ET, Park J-W, Kwak J-H, Lee J-O, Gu MB. 2011. Conductive quantum dot-encapsulated electrospun nanofibers from polystyrene and polystyrene-co-maleic anhydride copolymer blend as gas sensors. React. Funct. Polym. 71: 104-108.
Urrutia P, Mateo C, Guisan JM, Wilson L, Illanes A. 2013. Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation. Biochem. Eng. J. 77: 41-48.
Van de Voorde I, Goiris K, Syryn E, Van den Bussche C, Aerts G. 2014. Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of D -tagatose production. Process Biochem. 49: 2134-2140.
Verma ML, Barrow CJ, Kennedy JF, Puri M. 2012. Immobilization of β- D -galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: characterization and lactose hydrolysis. Int. J. Biol. Macromol. 50: 432-437.
Vieira DC, Lima LN, Mendes AA, Adriano WS, Giordano RC, Giordano RLC, Tardioli PW. 2013. Hydrolysis of lactose in whole milk catalyzed by β-galactosidase from Kluyveromyces fragilis immobilized on chitosan-based matrix. Biochem. Eng. J. 81: 54-64.
Wong DE, Dai M, Talbert JN, Nugen SR, Goddard JM. 2014. Biocatalytic polymer nanofibers for stabilization and delivery of enzymes. J. Mol. Catal. B Enzym. 110: 16-22.
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