A Simple Method for Beta-glucosidase Immobilization and Its Application in Soybean Isoflavone Glycosides Hydrolysis

다국어 초록 (Multilingual Abstract)

In this study, a simple, inexpensive and fast β-glucosidase immobilization system was constructed and evaluated in isoflavone glycosides hydrolysis. A β-glucosidase gene from Thermoascus aurantiacus IFO9748 was recombinantly expressed in Pichia pastoris KM71H and immobilized on regenerated amorphous cellulose (RAC) by fused cellulose binding module 3. Through simple mixing cellulose and crude enzyme for 15 min under room temperature, 96.04% β-glucosidase was immobilized onto RAC. The optimum temperature for β-glucosidase activity was increased by 5ºC after immobilization. The half-life (t½) of heat inactivation of immobilized enzyme at 60oC was improved over 8 folds. After 30 rounds recycled at 40oC, 96.9% daidzin and 98.9% genistin could still be hydrolyzed. A continuous hydrolysis system was also constructed, and at the flow rate of 0.2 mL/min after 30 h hydrolysis, 95.6% genistin and 90.2% daidzin can still be hydrolyzed. Combined the simple and high efficient enzyme immobilization procedure and inexpensive cellulose, this scalable and practical system may have broad prospects for industrial utilization.

참고문헌 (Reference)

1 Rostagno, M. A., "Ultrasoundassisted extraction of soy isoflavones" 1012 : 119-128, 2003

2 Chang, M. Y., "Thermal inactivation and reactivity of beta-glucosidase immobilized on chitosan-clay composite" 43 : 48-53, 2008

3 Jana, A., "Tannase immobilization by chitin-alginate based adsorption-entrapment technique and its exploitation in fruit juice clarification" 8 : 2319-2329, 2015

4 Shinjiro, I., "Soybean and processed soy foods ingredients, and their role in cardiometabolic risk prevention" 7 : 75-82, 2015

5 Izumi, T., "Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans" 130 : 1695-1699, 2000

6 Murashima, K., "Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase EngD from Clostridium cellulovorans" 50 : 620-628, 2003

7 Hong, J., "Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage" 1194 : 150-154, 2008

8 Verma, M. L., "Recent trends in nanomaterials immobilised enzymes for biofuel production" 36 : 108-119, 2016

9 Karav, S., "Recent advances in immobilization strategies for glycosidases" 33 : 104-112, 2017

10 Hong, J., "Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications" 23 : 12535-12540, 2007

11 Gueguen, Y., "Purification and characterization of an intracellular betaglucosidase from Botrytis cinerea" 17 : 900-906, 1995

12 Tran, D. N., "Perspective of recent progress in immobilization of enzymes" 1 : 956-968, 2011

13 Chiang, W. D., "Optimization of acid hydrolysis conditions for total isoflavones analysis in soybean hypocotyls by using RSM" 72 : 499-503, 2001

14 Carvalho, Y., "Nanosilicalites as support for beta-glucosidases covalent immobilization" 182 : 1619-1629, 2017

15 Ye, P., "Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization" 27 : 4169-4176, 2006

16 Levitsky, V. Y., "Kinetic analysis of deactivation of immobilized alpha-chymotrypsin by water-miscible organic solvent in kyotorphin synthesis" 65 : 170-175, 1999

17 Yatsu, F. K. J., "Isoflavone-aglycone fraction from Glycine max: a promising raw material for isoflavone-based pharmaceutical or nutraceutical products" 26 : 259-267, 2016

18 Tischer, W., "Immobilized enzymes: Methods and applications" 200 : 95-126, 1999

19 Romo-Sanchez, S., "Immobilization of betaglucosidase and its application for enhancement of aroma precursors in muscat wine" 7 : 1381-1392, 2014

20 Tu, M. B., "Immobilization of beta-glucosidase on Eupergit C for lignocellulose hydrolysis" 28 : 151-156, 2006

21 Su, E. Z., "Immobilization of beta-glucosidase and its aroma-increasing effect on tea beverage" 88 : 83-89, 2010

22 Gurdas, S., "Immobilization of Aspergillus oryzae beta-galactosidase onto duolite A568 resin via simple adsorption mechanism" 5 : 904-911, 2012

23 Choi, Y. B., "Hydrolysis of soybean isoflavone glucosides by lactic acid bacteria" 24 : 2113-2116, 2002

24 Chen, K. I., "Hydrolysis of isoflavone in black soy milk using cellulose bead as enzyme immobilizer" 24 : 788-795, 2016

25 Chang, J., "Hydrolysis of isoflavone glycoside by immobilization of beta-glucosidase on a chitosan-carbon in two-phase system" 61 : 465-470, 2013

26 Pei, X., "Heterologous expression of a GH3 beta-glucosidase from Neurospora crassa in Pichia pastoris with high purity and its application in the hydrolysis of soybean isoflavone glycosides" 119 : 75-84, 2016

27 Khan, M., "Graphene based magnetic nanocomposites as versatile carriers for high yield immobilization and stabilization of beta-galactosidase" 6 : 53493-53503, 2016

28 Warmerdam, A., "Galacto-oligosaccharide production with immobilized beta-galactosidase in a packed-bed reactor vs. free beta-galactosidase in a batch reactor" 92 : 383-392, 2014

29 Liggins, J., "Extraction and quantification of daidzein and genistein in food" 264 : 1-7, 1998

30 Richins, R. D., "Expression, immobilization, and enzymatic characterization of cellulosebinding domain-organophosphorus hydrolase fusion enzymes" 69 : 591-596, 2000

31 Wan, W., "Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast" 91 : 789-798, 2011

32 Chen, K. I., "Enrichment of two isoflavone aglycones in black soymilk by using spent coffee grounds as an immobiliser for beta-glucosidase" 139 : 79-85, 2013

33 Chen, K. I., "Enrichment of two isoflavone aglycones in black soymilk by immobilized beta-glucosidase on solid carriers" 60 : 12540-12546, 2012

34 Ahn, J. O., "Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain" 64 : 833-839, 2004

35 Taku, K., "Effect of soy isoflavone extract supplements on bone mineral density in menopausal women: meta-analysis of randomized controlled trials" 19 : 33-42, 2010

36 Delmonte, P., "Determination of isoflavones in dietary supplements containing soy, red clover and kudzu: Extraction followed by basic or acid hydrolysis" 1107 : 59-69, 2006

37 Alftren, J., "Covalent immobilization of beta-glucosidase on magnetic particles for lignocellulose hydrolysis" 169 : 2076-2087, 2013

38 Rodriguez-Colinas, B., "Continuous packed bed reactor with immobilized beta-galactosidase for production of galactooligosaccharides (GOS)" 6 : 189-, 2016

39 Song, X. F., "Comparison of three thermostable beta-glucosidases for application in the hydrolysis of soybean isoflavone glycosides" 59 : 1954-1961, 2011

40 Hong, J., "Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast" 63 : 42-50, 2003

41 Hong, J., "Cloning and functional expression of thermostable beta-glucosidase gene from Thermoascus aurantiacus" 73 : 1331-1339, 2007

42 Celik, A., "Characterization of beta-glucosidase immobilized on chitosan-multiwalled carbon nanotubes (MWCNTS) and their application on tea extracts for aroma enhancement" 89 : 406-414, 2016

43 Fang, W., "Characterization of a novel beta-glucosidase from Gongronella sp W5 and its application in the hydrolysis of soybean isoflavone glycosides" 62 : 11688-11695, 2014

44 Grigoras, A. G., "Catalase immobilization-A review" 117 : 1-20, 2017

45 Ahmed, S. A., "Biochemical studies on immobilized fungal beta-glucosidase" 30 : 747-758, 2013

46 Sheldon, R. A., "Biocatalysis engineering: the big picture" 46 : 2678-2691, 2017

47 Mohamad, N. R., "An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes" 29 : 205-220, 2015

48 Ye, P., "Adsorption and activity of lipase from Candida rugosa on the chitosan-modified poly (acrylonitrile-co-maleic acid) membrane surface" 60 : 62-67, 2007