为了获取具有广泛适用性的DNA聚合酶,将深海热液区获得的超嗜热古菌Palaeococcus pacificus DY20341的Pol B DNA聚合酶和d UTPase进行原核表达和纯化,将得到的Pol B DNA聚合酶及d UTPase混合后用于扩增古菌、细菌和真核生物的基因.通过...为了获取具有广泛适用性的DNA聚合酶,将深海热液区获得的超嗜热古菌Palaeococcus pacificus DY20341的Pol B DNA聚合酶和d UTPase进行原核表达和纯化,将得到的Pol B DNA聚合酶及d UTPase混合后用于扩增古菌、细菌和真核生物的基因.通过PCR反应成功地扩增了商业化Pfu DNA聚合酶所不能扩增的Palaeococcus pacificus DY2034的琼脂酶基因.由此可知,尽管商业化的DNA聚合酶多种多样,针对不同扩增目的片段所需的特异DNA聚合酶体系仍然是必要的,这是又一嗜热古菌B家族DNA聚合酶应用的报道.展开更多
By heterologous expression of a gene from Palaeococcus ferrophilus,a novel recombinant enzyme designated AMPf was obtained and proved to be an amylolytic enzyme with unique catalytic characteristics.The optimal temper...By heterologous expression of a gene from Palaeococcus ferrophilus,a novel recombinant enzyme designated AMPf was obtained and proved to be an amylolytic enzyme with unique catalytic characteristics.The optimal temperature and pH of AMPf were 50℃ and 7.0 respectively.Although sequence analysis and acarbose hydrolysis ability indicated that AMPf belongs to the subfamily GH13_20,interestingly,this enzyme hardly acts on cyclodextrins and pullulan distinguishing it from most enzymes in this subfamily.AMPf hydrolyzes starches to glucose,maltose,maltotriose,and maltotetrose as main products.AMPf mainly liberates glucose from starch with the concentration of 1%(w/v),while it shows malto-oligosaccharide forming ability with higher starch concentration of 4%(w/v).Also,the 4,6-ethylidene-4-nitrophenyl-maltoheptaose hydrolysis ability further indicates the unique combination endo-acting and glucose releasing exo-acting activtity of AMPf.AMPf could utilize maltose and maltotriose to produce malto-oligosaccharides by transglycosylation activity.It was proven AMPf has application protential in malto-oligosaccharides production.展开更多
基金financially supported by National Natural Science Foundation of China(32072268)The Science&Technology Pillar Program of Jiangsu Province(BE2018304)+3 种基金National First class Discipline Program of Food Science and Technology(JUFSTR20180203)National Natural Science Foundation of China(32072164)National Key Research and Development Program of China(2020YFC1606804)National Natural Science Foundation of China(32101990).
文摘By heterologous expression of a gene from Palaeococcus ferrophilus,a novel recombinant enzyme designated AMPf was obtained and proved to be an amylolytic enzyme with unique catalytic characteristics.The optimal temperature and pH of AMPf were 50℃ and 7.0 respectively.Although sequence analysis and acarbose hydrolysis ability indicated that AMPf belongs to the subfamily GH13_20,interestingly,this enzyme hardly acts on cyclodextrins and pullulan distinguishing it from most enzymes in this subfamily.AMPf hydrolyzes starches to glucose,maltose,maltotriose,and maltotetrose as main products.AMPf mainly liberates glucose from starch with the concentration of 1%(w/v),while it shows malto-oligosaccharide forming ability with higher starch concentration of 4%(w/v).Also,the 4,6-ethylidene-4-nitrophenyl-maltoheptaose hydrolysis ability further indicates the unique combination endo-acting and glucose releasing exo-acting activtity of AMPf.AMPf could utilize maltose and maltotriose to produce malto-oligosaccharides by transglycosylation activity.It was proven AMPf has application protential in malto-oligosaccharides production.
