多孔化CLEAs催化降解壳聚糖制备活性低聚糖
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摘要
针对传统交联酶聚体(CLEAs)催化大分子底物传质限制严重的问题,作者利用多孔化CLEAs(p-CLEAs)实现了壳聚糖酶法降解制备生物活性低聚糖。相同条件下p-CLEAs催化所生成还原糖量是游离酶的85%,是传统CLEAs的4倍。p-CLEAs所具有的多孔化结构造成了反应物相对分子质量和多分散性指数具有与游离酶催化不同的变化规律。通过控制酶解时间,结合超滤膜分离得到了相对分子质量为5 000~10 000的低聚糖产品,占总酶解产物质量的67.5%。抑菌性实验发现,当低聚壳聚糖(5 000~10 000)质量浓度为0.275 mg/m L时,对金黄色葡萄球菌和大肠杆菌达到完全抑制。
In order to overcome the defect of low catalytic efficiency of conventional cross-linked enzyme aggregates(CLEAs) toward macromolecular substrate,porous CLEAs(p-CLEAs) was applied in the degradation of chitosan to produce bioactive chitosan oligomers. The reducing sugar concentration was 85% of that catalyzed by free papain,which is 4 times higher than the conventional CLEAs under the same condition. Molecular weight and polydispersity index displayed the particular properties in the degradation of chitosan catalyzed by p-CLEAs compared with free papain. By controlling the degradation time,67.5%(wt) of the oligomer product(5~10 k D) were obtained through ultrafiltration membrane separation. Staphylococcus aureus and Escherichia coli can be totally suppressed when the concentration of oligomers(5~10 k D) was 0.275 mg/m L.
In order to overcome the defect of low catalytic efficiency of conventional cross-linked enzyme aggregates(CLEAs) toward macromolecular substrate,porous CLEAs(p-CLEAs) was applied in the degradation of chitosan to produce bioactive chitosan oligomers. The reducing sugar concentration was 85% of that catalyzed by free papain,which is 4 times higher than the conventional CLEAs under the same condition. Molecular weight and polydispersity index displayed the particular properties in the degradation of chitosan catalyzed by p-CLEAs compared with free papain. By controlling the degradation time,67.5%(wt) of the oligomer product(5~10 k D) were obtained through ultrafiltration membrane separation. Staphylococcus aureus and Escherichia coli can be totally suppressed when the concentration of oligomers(5~10 k D) was 0.275 mg/m L.
引文
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[20]TANDJAOUI N,TASSIST A,ABOUSEOUD M,et al.Preparation and characterization of cross-linked enzyme aggregates(CLEAs)of Brassica rapa peroxidase[J].Biocatalysis&Agricultural Biotechnology,2015,4(2):208-213.
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[23]DANIEL I P,FRED V R,ROGER A S.Cross-linked enzyme aggregates of chloroperoxidase:synthesis,optimization and characterization[J].Advanced Synthesis&Catalysis,2009,351(351):2133-2139.
[24]MONTORO-G S,GIL O F,NAVARRO F J,et al.Improved cross-linked enzyme aggregates for the production of desacetyl beta-lactam antibiotics intermediates[J].Bioresource Technology,2010,101(1):331-336.
[25]WANG M,JIA C,QI W,et al.Porous-CLEAs of papain:application to enzymatic hydrolysis of macromolecules[J].Bioresource Technology,2011,102(3):3541-3545.
[26]OKAMOTO K,KITANO H,AKAZAWA T.Biosynthesis and excretion of hydrolases in germinating cereal seeds[J].Plant and Cell Physiology,1980,21(1):201-204.
[27]IMOTO T K Y.A simple activity measurement of lysozyme[J].Agricultural and Biological Chemistry,1971,35:1154-1156.
[28]LIU Yijun,JIANG Ying,FENG Yunfang,et al.The study of molecular weight and distribution in the process of oxidative degradation of chitosan by GPC[J].Journal of Functional Polymer,2004,17(4):671-674.(in Chinese)
[2]KHOR E,LIM L Y.Implantable applications of chitin and chitosan[J].Biomaterials,2003,24(13):2339-2349.
[3]MACLAUGHLIN F C,MUMPER R J,WANG J,et al.Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery[J].Journal of Controlled Release,1998,56(56):259-272.
[4]TOKURA S,UENO K,MIYAZAKI S,et al.Molecular weight dependent antimicrobial activity by Chitosan[J].Macromolecular Symposia,1997,120(1):1-9.
[5]KUMAR A B V,VARADARAJ M C,LALITHA R G,et al.Low molecular weight chitosans:preparation with the aid of papain and characterization[J].Biochimica Et Biophysica Acta,2004,1670(2):137-146.
[6]YIN Xuhong,SUN Tao,YAO Qian,et al.DPPH scavenging activity of chitosan oligosaccharide zinc complexes[J].Journal of Food Science and Biotechnology,2009,28(3):329-331.(in Chinese)
[7]JIA Z,SHEN D.Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid[J].Carbohydrate Polymers,2002,49(4):393-396.
[8]QIN C Q,DU Y M,XIAO L.Effect of hydrogen peroxide treatment on the molecular weight and structure of chitosan[J].Polymer Degradation&Stability,2002,76(2):211-218.
[9]CZECHOWSKA-BISKUP R,ROKITA B,LOTFY S,et al.Degradation of chitosan and starch by 360 k Hz ultrasound[J].Carbohydrate Polymers,2005,60(2):175-184.
[10]GE H,LUO D.Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation[J].Carbohydrate Research,2005,340(7):1351-1356.
[11]GUOJANE T,WU Z Y,SU W H.Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation[J].Journal of Food Protection,2000,63(6):747-752.
[12]SHARP R G.A review of the applications of chitin and its derivatives in agriculture to modify plant-microbial interactions and improve crop yields[J].Agronomy,2013,3(3):757-793.
[13]NAM K S,CHOI Y R,SHON Y H.Evaluation of the antimutagenic potential of chitosan oligosaccharide:Rec,Ames adn Umu tests[J].Biotechnology Letters,2001,23(23):971-975.
[14]NGO D N,QIAN Z J,JE J Y,et al.Aminoethyl chitooligosaccharides inhibit the activity of angiotensin converting enzyme[J].Process Biochemistry,2008,43(1):119-123.
[15]MATEI P M,MARTIN-RAMOS P,SANCHEZ-BASCONES M,et al.Synthesis of chitosan oligomers/propolis/silver-nanoparticles composite systems and study of their activity against diplodia seriata[J].International Journal of Polymer Science,2015,864729,1-11.
[16]LEE D X,XIA W S,ZHANG J L.Enzymatic preparation of chitooligosaccharides by commercial lipase[J].Food Chemistry,2008,111(2):291-295.
[17]LI J,DU Y,YANG J,et al.Preparation and characterisation of low molecular weight chitosan and chito-oligomers by a commercial enzyme[J].Polymer Degradation&Stability,2005,87(3):441-448.
[18]RAMIREZ-COUTINO L,REVAH S,SHIRAI K.Enzymatic hydrolysis of chitin in the production of oligosaccharides using Lecanicillium fungicola chitinases[J].Process Biochemistry,2006,41(5):1106-1110.
[19]SHELDON R A.Enzyme immobilization:the quest for optimum performance[J].Advanced Synthesis&Catalysis,2007,38(36):1289-1307.
[20]TANDJAOUI N,TASSIST A,ABOUSEOUD M,et al.Preparation and characterization of cross-linked enzyme aggregates(CLEAs)of Brassica rapa peroxidase[J].Biocatalysis&Agricultural Biotechnology,2015,4(2):208-213.
[21]AYTAR B S,BAKIR U.Preparation of cross-linked tyrosinase aggregates[J].Process Biochemistry,2008,43(2):125-131.
[22]LANGEN L M,Van,SELASSA R P,FRED V R,et al.Cross-linked aggregates of(R)-oxynitrilase:a stable,recyclable biocatalyst for enantioselective hydrocyanation[J].Organic Letters,2005,7(2):327-329.
[23]DANIEL I P,FRED V R,ROGER A S.Cross-linked enzyme aggregates of chloroperoxidase:synthesis,optimization and characterization[J].Advanced Synthesis&Catalysis,2009,351(351):2133-2139.
[24]MONTORO-G S,GIL O F,NAVARRO F J,et al.Improved cross-linked enzyme aggregates for the production of desacetyl beta-lactam antibiotics intermediates[J].Bioresource Technology,2010,101(1):331-336.
[25]WANG M,JIA C,QI W,et al.Porous-CLEAs of papain:application to enzymatic hydrolysis of macromolecules[J].Bioresource Technology,2011,102(3):3541-3545.
[26]OKAMOTO K,KITANO H,AKAZAWA T.Biosynthesis and excretion of hydrolases in germinating cereal seeds[J].Plant and Cell Physiology,1980,21(1):201-204.
[27]IMOTO T K Y.A simple activity measurement of lysozyme[J].Agricultural and Biological Chemistry,1971,35:1154-1156.
[28]LIU Yijun,JIANG Ying,FENG Yunfang,et al.The study of molecular weight and distribution in the process of oxidative degradation of chitosan by GPC[J].Journal of Functional Polymer,2004,17(4):671-674.(in Chinese)