从嗜酸普鲁兰芽孢杆菌基因组中扩增出普鲁兰酶基因PulA，并将该基因连接到大肠杆菌表达载体pET-28a中，构建了普鲁兰酶基因的诱导表达载体pET-28a-PulA。测序结果表明，普鲁兰酶基因PulA长度为2766bp，编码922个氨基酸。将重组载体转化至大肠杆菌BL21（DE3）后，普鲁兰酶基因PulA在IPTG诱导下获得表达，产生胞内蛋白。SDS-PAGE测定的分子量约为110kD。细胞超声破碎液酶活为0.45U/mL。该酶的最适温度为55℃，最适pH为5.0，金属离子对酶活性影响不显著，具有I型普鲁兰酶特性。此重组普鲁兰酶的酶学性质表明此酶具有独特的耐酸性质，具备一定的工业化应用价值。
Abstract
The pullulanase gene from the Bacillus acidopullulyticus genome has been obtained by PCR amplification. By connecting the gene to the inducible expression plasmid of pET-28a, a pullulanase gene inducible expression plasmid of pET-28a-PulA was constructed. The pullulanase gene was 2766bp in length and encoded 922 amino acids. The competent cells of E. coli BL21 (DE3) were transformed with the recombinant plasmid, and after inducement by IPTG, the pullulanase was able to be expressed and examined from the inside of the cells. The enzyme activity of the recombinant pullulanase obtained from ultrasonic broken cells was 0.45U/mL. SDS-PAGE analysis showed that the molecular weight of the protein was approximately 110kD. The maximum activity of the enzyme occurred at 55℃ and pH 5.0. Metal ions had no significant influence on the enzyme activity. The enzyme belongs to the type I pullulanase class. The recombinant pullulanase showed excellent acid resistance so that it may be useful for applications of pullulanase in industry.
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
［1］Ling H S, Ling T C, Mohamad R, et al. Characterization of pullulanase type Ⅱ from Bacillus cereus H1.5[J]. American Journal of Biochemistry and Biotechnology, 2009, 5(4): 170-179.
[2］Kang J, Park K-M, Choi K-H, et al. Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana[J]. Enzyme and Microbial Technology, 2011, 48(3): 260-266.
[3］Jiao Y L, Wang S J, Lv M S, et al. A GH57 family amyjopullulanase from deep-sea thermococcus siculi: expression of the gene and characterization of the recombinant enzyme[J]. Current Microbiology, 2011, 62: 222-228.
[4］Wallenfel K, Bender H, Rached J R. Pullulanase from Aerobacter aerogenes: production in a cell-bound state. Purification and properties of the enzyme[J]. Biochemical and Biophysical Research Communications, 1966, 22(3): 254-261.
[5］Costanzo B, Garabed A. Starchhydrolyzing enzymes from thermophilic archaea and bacteria[J]. Current Opinion in Chemical Biology, 2002, 6(2): 151-160.
[6］Niehaus F, Bertoldo C, Kahler M, et al. Extremophiles as a source of novel enzymes for industrial application[J]. Applied Microbiology and Biotechnology, 1999, 51(6): 711-729.
[7］Turkenburg J P, Brzozowski A M, Svendsen A, et al. Structure of a pullulanase from Bacillus acidopullulyticus[J]. Proteins, 2009, 76(2): 516-519. [8]Malviya S N, Malakar R, Tiwari A. Pullulanase: a potential enzyme for industrial application[J]. International Journal of Materials Research, 2010, 1(2): 10-20.
[9］Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227(5259): 680-685.
[10］乔宇, 丁宏标, 闫俊艳, 等. 一株普鲁兰酶产生菌的分离鉴定及发酵条件的优化[J]. 中国农业科技导报, 2012, 14(4): 81-86.
Qiao Y, Ding H B, Yan J Y, et al. Isolation, identification of pullulanase producing strain and optimization of its fermentation conditions[J]. Journal of Agricultural Science and Technology, 2012, 14(4): 81-86. (in Chinese)
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
PDF(1659 KB)
