磁性松香基高分子固定化交联脂肪酶聚集体的制备及性质研究
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摘要
制备了磁性松香基高分子固定化交联脂肪酶聚集体(固定化CLEAs-L)。研究制备条件对固定化CLEAs-L活性的影响,并研究了固定化CLEAs-L的结构与性质。结果表明,固定化CLEAs-L的最佳制备条件为:沉淀剂无水乙醇用量30%,脂肪酶质量浓度4 g/L,一次交联反应中添加0. 3%的戊二醛,交联反应2 h,二次交联反应中添加1%的戊二醛,交联脂肪酶聚集体与载体的质量比2. 5∶1。在最佳条件下,固定化CLEAs-L的酶活回收率为86. 52%,固定化CLEAs-L的最适温度和pH分别为45℃和7. 0。与游离脂肪酶、CLEAs-L相比,固定化CLEAs-L热稳定性和储存稳定性明显提高;重复操作6次后,固定化CLEAs-L的酶活回收率仍保持在60. 00%以上。
The immobilized cross-linked enzyme aggregates (CLEAs-L) on magnetic rosin-based polymer were prepared. The effects of preparation conditions on the activity of immobilized CLEAs-L were evaluated. Moreover,the properties and structures of immobilized CLEAs-L were investigated.The results showed that the optimal preparation conditions were obtained as follows: dosage of precipitant (absolute ethanol) 30%,lipase mass concentration 4 g/L,glutaraldehyde dosage 0. 3% in the first cross-linking reaction,reaction time 2 h,glutaraldehyde dosage 1% in the second cross-linking reaction,and the mass ratio of CLEAs-L to carrier 2. 5∶ 1. Under these conditions,the recovery rate of enzyme activity was 86. 52%,and the optimal temperature and pH of immobilized CLEAs-L were 45 ℃and 7. 0 respectively. The thermal and storage stability of immobilized CLEAs-L were improved remarkably compared with the free lipase and CLEAs-L. After six batch reactions,the recovery rate of enzyme activity of immobilized CLEAs-L was still above 60. 00%.
The immobilized cross-linked enzyme aggregates (CLEAs-L) on magnetic rosin-based polymer were prepared. The effects of preparation conditions on the activity of immobilized CLEAs-L were evaluated. Moreover,the properties and structures of immobilized CLEAs-L were investigated.The results showed that the optimal preparation conditions were obtained as follows: dosage of precipitant (absolute ethanol) 30%,lipase mass concentration 4 g/L,glutaraldehyde dosage 0. 3% in the first cross-linking reaction,reaction time 2 h,glutaraldehyde dosage 1% in the second cross-linking reaction,and the mass ratio of CLEAs-L to carrier 2. 5∶ 1. Under these conditions,the recovery rate of enzyme activity was 86. 52%,and the optimal temperature and pH of immobilized CLEAs-L were 45 ℃and 7. 0 respectively. The thermal and storage stability of immobilized CLEAs-L were improved remarkably compared with the free lipase and CLEAs-L. After six batch reactions,the recovery rate of enzyme activity of immobilized CLEAs-L was still above 60. 00%.
引文
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[3]BADGUJAR V C,BADGUJAR K C,YEOLE P M,et al.Immobilization of Rhizomucor miehei lipase on a polymeric film for synthesis of important fatty acid esters:kinetics and application studies[J].Bioprocess Biosyst Eng,2017,40(10):1463-1478.
[4]崔建东,刘容麟.苯丙氨酸解氨酶印迹交联酶聚体的制备及部分催化性能研究[J].河北科技大学学报,2015,36(6):633-638.
[5]WAN L H,JIANG X L,LIU Y M,et al.Screening of lipase inhibitors from Scutellaria baicalensis extract using lipase immobilized on magnetic nanoparticles and study on the inhibitory mechanism[J].Anal Bioanal Chem,2016,408(9):2275-2283.
[6]CAO L,VAN LANGEN L,SHELDON R A.Immobilized enzymes:carrier-bound or carrier-free[J].Curr Opin Biotech,2003,14(4):387-394.
[7]LIMA L C D,PERES D G C,MENDES A A.Kinetic and thermodynamic studies on the enzymatic synthesis of wax ester catalyzed by lipase immobilized on glutaraldehyde-activated rice husk particles[J].Bioprocess Biosyst Eng,2018,41:991-1002.
[8]BRAUN C,EWERTt J,STRESSLER T.Characterization of cross-linked enzyme aggregates(CLEAs)of the fusion protein FUS-Pep N_Pep X and their application for milk protein hydrolysis[J].Eur Food Res Technol,2017,243(10):1815-1828.
[9]王冬伟,孙谧.固载化木聚糖酶交联酶聚集体的制备及性质[J].中国海洋药物,2016,35(1):39-49.
[10]GAO J,SHI L L,JIANG Y J,et al.Formation of lipase Candida sp.99-125 CLEAs in mesoporous silica:characterization and catalytic properties[J].Catal Sci Technol,2013(3):3353-3359.
[11]ZEINALI M,LENJANNEZHADIAN H.Degradation of urea by entrapped cross-linked urease aggregates:a combinatorial approach to urease stabilization for environmental and industrial applications[J].Int J Environ Sci Technol,2018,15(1):49-56.
[12]黎克纯,雷福厚,卢建芳,等.松香基磁性微球的制备及其对Mn的吸附研究[J].林产化学与工业,2018,38(4):87-94.
[13]刘磊磊.磁性Fe3O4/PS基纳米材料的制备及其固定脂肪酶的研究[D].安徽芜湖:安徽工程大学,2017.
[14]陈颖,肖辰鹏,陈晓云,等.交联海藻糖合酶聚集体的制备及性质[J].食品科学,2013,34(9):108-113.
[15]BONAZZA H,MANZO R M,SANTOS D,et al.Operational and thermal stability analysis of Thermomyces lanuginosus lipase covalently immobilized onto modified chitosan supports[J].Appl Biochem Biotech,2018,184(1):182-196.
[16]黎克纯,卢建芳,周菊英,等.具有菲环骨架的高分子载体固定淀粉酶交联聚集体[J].食品科学,2017,38(14):112-119.
[17]WANG M,QI W,YU Q,et al.Cross-linking enzyme aggregates in the macropores of silica gel:a practical and efficient method for enzyme stabilization[J].Biochem Eng J,2010,52(2):168-174.