碱性蛋白酶交联聚集体的制备及其催化性能研究
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摘要
碱性蛋白酶在食品、医药、酿造、丝绸、皮革等行业中发挥着重要作用。制备了碱性蛋白酶交联体,并对其催化性能进行研究。在最佳制备条件下(90%叔丁醇作为沉淀剂,沉淀时间为15min,交联剂浓度为33 mmol/L,交联时间为6 h),交联酶的酶活回收率为22. 6%。与游离酶相比,交联酶的最适pH值向碱性方向变化,由7. 5变为8. 0,最适温度由60℃变成65℃。酶动力学研究表明,交联酶对酪蛋白的催化水解能力(4. 3 min~(-1))比游离酶(3. 7 min~(-1))更高。尽管交联酶最大反应速度V_(max)(9. 8 mg/(m L·min))低于游离酶(13. 3 mg/(m L·min)),但交联酶对底物的亲和力Km(2. 3 mg/m L)比游离酶(3. 6 mg/m L)有所增加,而且其热稳定性和酸碱稳定性都得到一定程度的提高。另外,在磷酸盐缓冲液中重复使用5和8批次后,交联酶还能保持82. 5%和56. 5%的酶活性。
Cross-linked enzyme aggregates( CLEAs) of alkaline protease play a crucial role in food,medicine, brewing, silk, leather, and other industries CLEAs were successfully prepared and characterized in this paper. Under the optimum preparation conditions( 90% tert-butanol as the precipitant,precipitated time of 15 min,glutaraldehyde concentration of 33 mmol/L and cross-linking time of 6 h),the activity recovery of CLEAs recorded 22. 6%. In addition,the CLEAs displayed a shift in optimal pH towards the alkaline side from 7. 5 to 8. 0,and their optimal temperature was also improved to a certain extent compared to free enzyme from 60 ℃ to 65 ℃. The enzymatic kinetics studies indicated that the CLEAs( 4. 3 min~(-1)) were more efficient than the free enzyme( 3. 7 min~(-1)) in catalyzing casein hydrolysis. Although the V_(max) of CLEAs( 9. 8 mg/( m L·min)) was lower than that of free enzyme( 13. 3 mg/( m L·min)),the substrate affinity of CLEAs( 2. 3 mg/m L) increased compared with the free enzyme( 3. 6 mg/m L). The CLEAs also enhanced the thermal and pH stability of alkaline protease. Moreover,after being used repeatedly for 5 and 8 batches in phosphate buffer,CLEAs retained 82. 5% and 56. 5%of their initial activity.
Cross-linked enzyme aggregates( CLEAs) of alkaline protease play a crucial role in food,medicine, brewing, silk, leather, and other industries CLEAs were successfully prepared and characterized in this paper. Under the optimum preparation conditions( 90% tert-butanol as the precipitant,precipitated time of 15 min,glutaraldehyde concentration of 33 mmol/L and cross-linking time of 6 h),the activity recovery of CLEAs recorded 22. 6%. In addition,the CLEAs displayed a shift in optimal pH towards the alkaline side from 7. 5 to 8. 0,and their optimal temperature was also improved to a certain extent compared to free enzyme from 60 ℃ to 65 ℃. The enzymatic kinetics studies indicated that the CLEAs( 4. 3 min~(-1)) were more efficient than the free enzyme( 3. 7 min~(-1)) in catalyzing casein hydrolysis. Although the V_(max) of CLEAs( 9. 8 mg/( m L·min)) was lower than that of free enzyme( 13. 3 mg/( m L·min)),the substrate affinity of CLEAs( 2. 3 mg/m L) increased compared with the free enzyme( 3. 6 mg/m L). The CLEAs also enhanced the thermal and pH stability of alkaline protease. Moreover,after being used repeatedly for 5 and 8 batches in phosphate buffer,CLEAs retained 82. 5% and 56. 5%of their initial activity.
引文
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[12]姜艳军,王旗,王温琴,等.交联酶聚集体与仿生硅化技术结合制备固定化脂肪酶[J].催化学报,2012,33:857-862.JIANG Y J,WANG Q,WANG W Q,et al. Preparation of immobilized lipase through combination of crosslinked enzyme aggregates and biomimetic silicification[J]. Chinese Journal of Catalysis,2012,33:857-862.
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[14] PANWAR D,KAIRA G S,KAPOOR M. Cross-linked enzyme aggregates(CLEAs)and magnetic nanocomposite grafted CLEAs of GH26 endo-β-1,4-mannanase:improved activity,stability and reusability[J]. International Journal of Biological Macromolecules,2017,105:1289-1299.
[15] SCHOEVAART R,WOLBERS M W,GOLUBOVIC M,et al. Preparation,optimization,and structures of crosslinked enzyme aggregates(CLEAs)[J]. Biotechnology&Bioengineering,2010,87:754-762.
[16] SHELDON R A. Characteristic features and biotechnological applications of cross-linked enzyme aggregates(CLEAs)[J]. Applied Microbiology&Biotechnology,2011,92:467-477.
[17] YU C Y,LI X F,LOU W Y,et al. Cross-linked enzyme aggregates of mung bean epoxide hydrolases:a highly active,stable and recyclable biocatalyst for asymmetric hydrolysis of epoxides[J]. Journal of Biotechnology,2013,166:12-19.
[18]陈翠翠,何腊平,郑佳,等.交联酶聚集体性能改善研究进展[J].食品工业科技,2018,39:304-309.CHEN C C,HE L P,ZHENG J,et al. Research progress of cross-linked enzyme aggregates improvement[J]. Science and Technology of Food Industry,2018,39:304-309.
[19] SUN W Q,DAVIDSON P. Protein inactivation in amorphous sucrose and protein inactivation in amorphous sucrose and trehalose matrices:effects of phase separation and crystallization[J]. Biochimica et Biophysica Acta(BBA)-General Subjects,1998,1425:235-244.
[20] LOWRY O H,ROSEBROUGH N J,FARR A L,et al.Protein measurement with the Folin phenol reagent[J].Journal of Biological Chemistry,1951,193:265-275.
[21] REHMAN S,BHATTI H N,BILAL M,et al. Crosslinked enzyme aggregates(CLEAs)of Pencilluim notatum lipase enzyme with improved activity,stability and reusability characteristics[J]. International Journal of Biological Macromolecules,2016,91:1161-1169.
[22] TALEKAR S,GHODAKE V,GHOTAGE T,et al.Novel magnetic cross-linked enzyme aggregates(magnetic CLEAs)ofα-amylase[J]. Bioresource Technology,2012,123:542-547.
[23] WANG M F,JIA C X,QI W,et al. Porous-CLEAs of papain:application to enzymatic hydrolysis of macromolecules[J]. Bioresource Technology,2011,102:3541-3545.
[24] WANG S N,ZHANG C R,QI B K,et al. Immobilized alcalase alkaline protease on the magnetic chitosan nanoparticles used for soy protein isolate hydrolysis[J].European Food Research&Technology,2014,239:1051-1059.
[25]SULEK F,FERNANDEZ D P,KNEZ Z,et al. Immobilization of horseradish peroxidase as crosslinked enzyme aggregates(CLEAs)[J]. Process Biochemistry,2011,46:765-769.
[26] AYTAR B S,BAKIR U. Preparation of cross-linked tyrosinase aggregates[J]. Process Biochemistry,2008,43:125-131.
[27] SANGEETHA K,ABRAHAM T E. Preparation and characterization of cross-linked enzyme aggregates(CLEA)of subtilisin for controlled release applications[J]. International Journal of Biological Macromolecules,2008,43:314-319.
[28] KAUL P,STOLZ A,BANERJEE U C. Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis[J]. Advanced Synthesis&Catalysis,2010,349:2167-2176.
[29] LEI H,WANG W,CHEN L L,et al. The preparation and catalytically active characterization of papain immobilized on magnetic composite microspheres[J].Enzyme&Microbial Technology,2004,35:15-21.
[30] ASGHER M,NOREEN S,BILAL M. Enhancement of catalytic,reusability,and long-term stability features of Trametes versicolor IBL-04 laccase immobilized on different polymers[J]. International Journal of Biological Macromolecules,2017,95:54-62.
[2] GUPTA R,BEG Q K,LORENZ P. Bacterial alkaline proteases:molecular approaches and industrial applications[J]. Applied Microbiology&Biotechnology,2002,59:15-32.
[3]邱并生.碱性蛋白酶[J].微生物学通报,2012,39:1859-1860.QIU B S. Alkaline Protease[J]. Microbiology China,2012,39:1859-1860.
[4]付瑞敏,蔺芳,陈五岭.一株耐盐性蛋白酶产生菌的诱变选育及酶学性质研究[J].中国调味品,2016,41(8):53-57.FU R M,LIN F,CHEN W L. Mutation breeding and enzymatic properties of a salt-tolerant protease-producing strain[J]. China Condiment,2016,41(8):53-57.
[5] DEMIR T,ONAL S. Bioaffinity immobilization and characterization ofα-galactosidase on aminophenylboronicacid derivatized chitosan and Sepabeads EC-EA[J].LWT-Food Science and Technology,2018,90:547-555.
[6] KIM H,CHOI N,OH S W,et al. Synthesis ofα-linolenic acid-rich triacylglycerol using a newly prepared immobilized lipase[J]. Food Chemistry,2017,237:654-658.
[7]杨景峰,罗志刚,罗发兴,等.不同载体材料在脲酶固定化中的应用[J].化学与生物工程,2007,24:55-57.YANG J F,LUO Z G,LUO F X,et al. Applications of different supports in immobilization of urease[J].Chemistry&Bioengineering,2007,24:55-57.
[8] CAO L,LANGEN L V,SHELDON R A. Immobilised enzymes:carrier-bound or carrier-free?[J]. Current Opinion in Biotechnology,2003,14:387-394.
[9] WANG M F,WEI Q,SU R X,et al. Advances in carrierbound and carrier-free immobilized nanobiocatalysts[J].Chemical Engineering Science,2015,135:21-32.
[10] ROESSL U,NAHCLLKA J,NIDETZKY B. Carrier-free immobilized enzymes for biocatalysis[J]. Biotechnology Letters,2010,32:341-350.
[11] KARTAL F,JANSSEN M H A,HOLLMANN F,et al.Improved esterification activity of Candida rugosa lipase in organic solvent by immobilization as cross-linked enzyme aggregates(CLEAs)[J]. Journal of Molecular Catalysis B:Enzymatic,2011,71:85-89.
[12]姜艳军,王旗,王温琴,等.交联酶聚集体与仿生硅化技术结合制备固定化脂肪酶[J].催化学报,2012,33:857-862.JIANG Y J,WANG Q,WANG W Q,et al. Preparation of immobilized lipase through combination of crosslinked enzyme aggregates and biomimetic silicification[J]. Chinese Journal of Catalysis,2012,33:857-862.
[13]罗建平,欧杰,潘利华.交联β-葡萄糖苷酶聚集体的制备及其性质[J].食品科学,2007,28:254-257.LUO J P,OU J,PAN L H. Preparation and properties of cross-linkedβ-glucosidase aggregates[J]. Food Science,2007,28:254-257.
[14] PANWAR D,KAIRA G S,KAPOOR M. Cross-linked enzyme aggregates(CLEAs)and magnetic nanocomposite grafted CLEAs of GH26 endo-β-1,4-mannanase:improved activity,stability and reusability[J]. International Journal of Biological Macromolecules,2017,105:1289-1299.
[15] SCHOEVAART R,WOLBERS M W,GOLUBOVIC M,et al. Preparation,optimization,and structures of crosslinked enzyme aggregates(CLEAs)[J]. Biotechnology&Bioengineering,2010,87:754-762.
[16] SHELDON R A. Characteristic features and biotechnological applications of cross-linked enzyme aggregates(CLEAs)[J]. Applied Microbiology&Biotechnology,2011,92:467-477.
[17] YU C Y,LI X F,LOU W Y,et al. Cross-linked enzyme aggregates of mung bean epoxide hydrolases:a highly active,stable and recyclable biocatalyst for asymmetric hydrolysis of epoxides[J]. Journal of Biotechnology,2013,166:12-19.
[18]陈翠翠,何腊平,郑佳,等.交联酶聚集体性能改善研究进展[J].食品工业科技,2018,39:304-309.CHEN C C,HE L P,ZHENG J,et al. Research progress of cross-linked enzyme aggregates improvement[J]. Science and Technology of Food Industry,2018,39:304-309.
[19] SUN W Q,DAVIDSON P. Protein inactivation in amorphous sucrose and protein inactivation in amorphous sucrose and trehalose matrices:effects of phase separation and crystallization[J]. Biochimica et Biophysica Acta(BBA)-General Subjects,1998,1425:235-244.
[20] LOWRY O H,ROSEBROUGH N J,FARR A L,et al.Protein measurement with the Folin phenol reagent[J].Journal of Biological Chemistry,1951,193:265-275.
[21] REHMAN S,BHATTI H N,BILAL M,et al. Crosslinked enzyme aggregates(CLEAs)of Pencilluim notatum lipase enzyme with improved activity,stability and reusability characteristics[J]. International Journal of Biological Macromolecules,2016,91:1161-1169.
[22] TALEKAR S,GHODAKE V,GHOTAGE T,et al.Novel magnetic cross-linked enzyme aggregates(magnetic CLEAs)ofα-amylase[J]. Bioresource Technology,2012,123:542-547.
[23] WANG M F,JIA C X,QI W,et al. Porous-CLEAs of papain:application to enzymatic hydrolysis of macromolecules[J]. Bioresource Technology,2011,102:3541-3545.
[24] WANG S N,ZHANG C R,QI B K,et al. Immobilized alcalase alkaline protease on the magnetic chitosan nanoparticles used for soy protein isolate hydrolysis[J].European Food Research&Technology,2014,239:1051-1059.
[25]SULEK F,FERNANDEZ D P,KNEZ Z,et al. Immobilization of horseradish peroxidase as crosslinked enzyme aggregates(CLEAs)[J]. Process Biochemistry,2011,46:765-769.
[26] AYTAR B S,BAKIR U. Preparation of cross-linked tyrosinase aggregates[J]. Process Biochemistry,2008,43:125-131.
[27] SANGEETHA K,ABRAHAM T E. Preparation and characterization of cross-linked enzyme aggregates(CLEA)of subtilisin for controlled release applications[J]. International Journal of Biological Macromolecules,2008,43:314-319.
[28] KAUL P,STOLZ A,BANERJEE U C. Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis[J]. Advanced Synthesis&Catalysis,2010,349:2167-2176.
[29] LEI H,WANG W,CHEN L L,et al. The preparation and catalytically active characterization of papain immobilized on magnetic composite microspheres[J].Enzyme&Microbial Technology,2004,35:15-21.
[30] ASGHER M,NOREEN S,BILAL M. Enhancement of catalytic,reusability,and long-term stability features of Trametes versicolor IBL-04 laccase immobilized on different polymers[J]. International Journal of Biological Macromolecules,2017,95:54-62.
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