为了使脂肪酶在固定化的过程中酶活力损失小的同时提高固定化脂肪酶的稳定性,对325目蛭石和1 000目蛭石进行酸改性,选取比表面积大、孔径结构特征丰富的改性后蛭石为载体,采用吸附-包埋法固定化脂肪酶,研究改性蛭石作为载体固定化脂肪酶的最优化条件及固定化脂肪酶的催化性能。分析表明,1 000目蛭石经过改性后比表面积比较大、孔径结构较丰富。脂肪酶的最优固定化工艺为:脂肪酶与载体的质量比为1∶1,吸附温度为25 ℃,吸附时间为3 h,海藻酸钠的质量浓度为20 g/L,明胶的质量浓度为5 g/L,CaCl2溶液的浓度为0.04 mol/L,在此条件下固定化脂肪酶的酶活力回收率可达到82.77%;固定化脂肪酶经过7次重复回收利用后,酶活力仍可保留原来的85.33%,说明其重复使用稳定性较好。
To reduce the loss of lipase activity during immobilization and improve the stability of immobilized lipase, 325-mesh and 1 000-mesh vermiculites were modified by acid. The modified vermiculite with large specific surface area and rich pore structure was selected as the carrier, and lipase was immobilized by the adsorption-embedding method. The optimal conditions for immobilizing lipase with modified vermiculite as the carrier and the catalytic activity of immobilized lipase were investigated. The results showed that the specific surface area was relatively large and the pore structure of vermiculite was relatively rich when 1 000-mesh vermiculite was used. The optimal immobilization conditions of lipase were as follows: the mass ratio of lipase to carrier was 1∶1; the adsorption temperature was 25 ℃; the adsorption time was 3 h; the concentration of sodium alginate was 20 g/L; and the concentration of gelatin was 5 g/L. Calcium chloride at 0.04 mol/L enhanced the recovery rate of the immobilized lipase to 87.27%; after 7 rounds of recycling, the enzymatic activity was 85.33% of that of the original, which indicated good stability of the enzyme.
[1] TURATI D F M, JUNIOR W G M, TERRASAN C R F,et al. Immobilization of lipase from penicillium sp. section gracilenta (CBMAI 1583) on different hydrophobic supports: Modulation of functional properties[J]. Molecules,2017,22(2):339-348.
[2] PEREIRA M G, VELASCO-LOZANO S, MORENO-PEREZ S,et al. Different covalent immobilizations modulate lipase activities of Hypocrea pseudokoningii[J].Molecules,2017,22(9):1 448-1 460.
[3] CHENHUI W, HAOBO H, WEI J,et al. Immobilization of thermostable lipase QLM on core-shell structured polydopamine-coated Fe3O4 nanoparticle [J]. Catalysts,2017,22(7):49-60.
[4] CESAR A G. New strategy for the immobilization of lipases on glyoxyl-agarose supports: Production of robust biocatalysts for natural oil transformation[J].Int J Mol Sci,2017,12(18):2 130-2 148.
[5] SHAN Z, JIE S,DENG Q C,et al. Preparation of carriers based on ZnO nanoparticles decorated on graphene oxide (GO) nanosheets for efficient immobilization of lipase from Candida rugosa[J].Molecules,2017,22(22):1 205-1 249.
[6] MALDONADO R R, LOPES D B, AGUIAR-OLIVEIRA E,et al. A review on geotrichum lipases: Production, purification, immobilization and applications[J].Chem Biochem Eng,2016,30(4):439-454.
[7] 唐雪娇,吴丰鹏,苗时雨,等.阴沟杆菌属产脂肪酶菌株的酶学特性、脂肪酶提取与固定化[J].南开大学学报(自然科学版),2013,43(2):12-16.
[8] 田运齐,吴小芳,侯文颖, 等.辣根过氧化物酶在MOF上的固定化研究[J].辽宁师范大学学报(自然科学版),2016,39(3):373-376.
[9] TIAN Y Q,WU X F,HOU W Y,et al.Immobilization of horseradish peroxiodase on the metal-organic framework[J]. Journal of Liaoning Normal University(Natural Science Edition), 2012,6(10): 138-148.
[10] JAKUB Z, KARINA S,AGMESEKA K R,et al. Immobilization of amano lipase onto stober silica surface: Process characterization and kinetic studies[J]. Open Chem, 2015,32(13): 138-148.
[11] 曹文娟,袁海生. 桦褶孔菌漆酶固定化及其对染料的降解[J].菌物学报,2016,35(3): 343-354.
[12] 宋辉,张文宇,王鹏举,等.L-谷氨酸氧化酶与CBD的融合表达及其在微晶纤维素上固定化分析[J].生物工程学报,2016, 32(10):1 348-1 361.
[13] NAZZOLY R, TIAGO L,ROCIO B C,et al. Reversible immobilization of lipases on heterofunctional octyl-amino agarose beads prevents enzyme desorption[J]. Molecules,2016,22(21):646-663.
[14] ALNOCH R C,MELO R R,PALOMO J M,et al. New tailor-made alkyl-aldehyde bifunctional supports for lipase immobilization[J]. Catalysts, 2016,6(12):191-193.
[15] MATTE C R, BORDIOHAO C, POPPE J K,et al. Physical-chemical properties of the support immobead 150 before and after the immobilization process of lipase[J]. Braz Chem Soc, 2017, 28(8):1 430-1 439.
[16] DE A, MORENO-PEREZ S,FERNÁNDE L G,et al. Immobilization of Moniliella spathulata R25L270 lipase on ionic, Hydrophobic and covalent supports: Functional properties and hydrolysis of sardine oil [J].Molecules,2017,22(10):1 508-1 520.
[17] 杨本宏,蔡敬民,吴克,等.海藻酸钠固定化根霉脂肪酶的制备及其性质[J].催化学报,2005,26(11):977-981.
[18] 王爱玲,杨江科,黄瑛,等.海藻酸钠明胶协同固定化黑曲霉脂肪酶[J].应用化工,2007,36(4):317-321;324.
[19] 王兰,曾琪静.改性蛭石负载 Fe、N共掺杂TiO2光催化剂的制备及可见光降解苯酚研究[J].化工新型材料,2016,44(9):107-109.
[20] 曾铃,付宏渊,李传常,等.面向能源与环境应用的蛭石结构修饰及其复合材料[J].硅酸盐学报,2016,44(8):1 226-1 234.
[21] 陈理想,吴平霄,杨林,等.有机改性蛭石的特性及其对Hg2+吸附性能的研究[J].环境科学学报,2015,35(4):1 054-1 060.
[22] WINKLER U K,STUCKMANN M. Glycogen,hyaluronate and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens[J].Journal of Bacteriology,1979, 138(3):663-670.
[23] MARTA Z B,DOROTA C D, SIÓDMIAK T, et al.Chitosan-collagen coated magnetic nanoparticles for Lipase immobilization-new type of “Enzyme Friendly” polymer Shell crosslinking with squaric acid [J]. Catalysts, 2017,7(1):26.
