A GH57 family amylopullulanase from deep-sea Thermococcus siculi: expression of the gene and characterization of the recombinant enzyme.
The gene encoding a new extracellular amylopullulanase (type II pullulanase) was cloned from an extremely thermophilic anaerobic archaeon Thermococcus siculi strain HJ21 isolated previously from a deep-sea hydrothermal vent. The functional hydrolytic domain of the amylopullulanase (TsiApuN) and its MalE fusion protein (MTsiApuN) were expressed heterologously. The complete amylopullulanase (TsiApu) was also purified from fermentation broth of the strain. The pullulanase and amylase activities of the three enzymes were characterized. TsiApu had optimum temperature of 95°C for the both activities, while MTsiApuN and TsiApuN had a higher optimum temperature of 100°C. The residual total activities of MTsiApuN and TsiApuN were both 89% after incubation at 100°C for 1 h, while that of TsiApu was 70%. For all the three enzymes the optimum pHs for amylase and pullulanase activities were 5.0 and 6.0, respectively. By analyzing enzymatic properties of the three enzymes, this study suggests that the carboxy terminal region of TsiApu might interfere with the thermoactivity. The acidic thermoactive amylopullulanases MTsiApuN and TsiApuN could be further employed for industrial saccharification of starch.
Jiao
Yu-Liang
YL
College of Marine Sciences, HuaiHai Institute of Technology, Lianyungang, 222005, People's Republic of China. laioni@126.com
Wang
Shu-Jun
SJ
Lv
Ming-Sheng
MS
Xu
Jin-Li
JL
Fang
Yao-Wei
YW
Liu
Shu
S
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D019641:DNA, Archaeal; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C082507:amylopullulanase