果聚糖蔗糖酶基因的克隆表达及酶学性质分析
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摘要
将来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的果聚糖蔗糖酶(levansucrase)基因进行克隆,并将其在Escherichia coli BL21(DE3)中表达。对发酵产酶后的菌体进行破壁和离心,粗酶液经镍柱纯化后,进行酶学性质研究。结果表明,重组酶的水解酶活和转移酶活最适温度分别为45℃和40℃,最适pH分别为6.5和6.0。此外,果聚糖蔗糖酶的K_m和V_(max)分别为150.74mmol/L和21.23μmol/(mL·min)。最后,在pH 6.0、40℃条件下,果聚糖蔗糖酶催化反应12 h后,体系中果聚糖质量分数可达34.05%。
Levansucrase from Bacillus amyloliquefaciens was cloned and heterologously expressed in Escherichia coli BL21(DE3). After purification,the enzymatic characterization of levansucrase was determined and analyzed. The optimum temperature of hydrolytic activity was 45 ℃ and the maximal transfructosylation activity was measured at 40 ℃. The optimum pH for hydrolytic and transfructosylation activities were 6.5 and 6.0,respectively. The K_m and V_(max) values for levansucrase were calculated to be 150. 74 mmol/L sucrose and 21. 23 μmol/( mL·min), respectively.Levansucrase-catalyzed reaction was investigated at pH 6.0 and 40 ℃. The result indicated that the content of levan was up to 34.05% in total products after 12 h.
Levansucrase from Bacillus amyloliquefaciens was cloned and heterologously expressed in Escherichia coli BL21(DE3). After purification,the enzymatic characterization of levansucrase was determined and analyzed. The optimum temperature of hydrolytic activity was 45 ℃ and the maximal transfructosylation activity was measured at 40 ℃. The optimum pH for hydrolytic and transfructosylation activities were 6.5 and 6.0,respectively. The K_m and V_(max) values for levansucrase were calculated to be 150. 74 mmol/L sucrose and 21. 23 μmol/( mL·min), respectively.Levansucrase-catalyzed reaction was investigated at pH 6.0 and 40 ℃. The result indicated that the content of levan was up to 34.05% in total products after 12 h.
引文
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[3]YOO S H,YOON E J,CHA J,et al.Antitumor activity of levan polysaccharides from selected microorganisms[J].International Journal of Biological Macromolecules,2004,34(1-2):37-41.
[4]SHIH I L,CHEN L D,WU J Y.Levan production using Bacillus subtilisnatto cells immobilized on alginate[J].Carbohydrate Polymers,2010(82):111-117.
[5]LU Juan,XIAO Min,LU Lili.Optimization of fermentation conditions for production of levan by Bacillus licheniformis 8-37-0-1[J].Chinese Journal of Food Science,2011(32):183-187.(in Chinese)
[6]ZHANG T,LI R,QIAN H,et al.Biosynthesis of levan by levansucrase from Bacillus methylotrophicus SK 21.002[J]Carbohydrate Polymers,2014(101):975-981.
[7]ESAWYA M A,AHMEDA E F,HELMYA W A,et al.Production of levansucrase from novel honey Bacillus subtilis isolates capable of producing antiviral levans[J].Carbohydrate Polymers,2011,86(2):823-830.
[8]KIM K H,CHUNG C B,KIM Y H,et al.Cosmeceutical properties of levan produced by Zymomonas mobilis[J].Cosmetic Science,2005,56(6):395-406.
[9]BEKERS M,LAUKEVICS J,UPITE D,et al.Fructooligosaccharide and levan producing activity of Zymomonas mobilis extracellular levansucrase[J].Process Biochemistry,2002,38(5):701-706.
[10]ABDEL-FATTAH A M,GAMAL-ELDEEN A M,HELMY W A,et al.Antitumor and antioxidant acivities of levan and its derivative from the isolate Bacillus subtilis NRC1aza[J].Carbohydrate Polymers,2012,89(2):314-322.
[11]OSCARSON S,SEHGELMEBLE F W.Chemical syntheses of inulin and levan structures[J].Journal of Organic Chemistry,2002,67(24):8457-8462.
[12]WANG Limei,ZHOU Huiming.Study on fermentation conditions for the production of fructosyltransferase and transfructosylation from Aspergillus niger[J].Journal of Food Science and Biotechnology,2006,25(2):53-54.(in Chinese)
[13]AMMAR Y B,MATSUBARA T,ITO K,et al.Characterization of a thermostable levansucrase from Bacillus sp.TH4-2 capable of producing high molecular weight levan at high temperature[J].Journal of Biotechnology,2002,99(2):111-119.
[14]BELGHITH H,SONG K B,KIM C H,et al.Optimal conditions for levan formation by an overexpressed recombinant levansucrase[J].Biotechnology Letters,1996,18(4):467-472.
[15]PARK H E,PARK N H,KIM M J,et al.Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from Microbacterium laevaniformans ATCC 15953[J].Enzyme and Microbial Technology,2003,32(7):820-827.
[16]TIEKING M,EHRMANN M A,VOGEL R F,et al.Molecular and functional characterization of a levansucrase from sourdough isolate Lactobacillus sanfranciscensis TMW 1.392[J].Applied Microbiology and Biotechnology,2005,66(6):655-663.
[17]KANG H K,SEO Y M,SEO E S,et al.Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli[J].Biochimica et Biophysica Acta,2005,1727(1):5-15.
[18]OHTSUKU K,HINO S,FUKUSHIMA T,et al.Characterization of levansucrase from Rahnella aquatilis JMC-1683[J]Bioscience Biotechnology and Biochemistry,1992,56(9):1373-1377.
[19]R.E.布坎南,N.E.吉本斯.伯杰细菌鉴定手册(第八版)[M].北京:科学出版社,1984.
[20]YANASE H,IWATA M,NAKAHIGASHI R,et al.Purification,crystallization and properties of the extracellular levansucrase from Zymomonas mobilis[J].Bioscience Biotechnology and Biochemistry,1992,56(8):1335-1336.