Cloning and expression of an Î±-amylase encoding gene from the hyperthermophilic archaebacterium<i>Thermococcus hydrothermalis</i>and biochemical characterisation of the recombinant enzyme

Lévêque, Emmanuel; Haye, Bernard; Belarbi, A.

doi:10.1111/j.1574-6968.2000.tb09083.x

Cited by 17 publications

(5 citation statements)

References 21 publications

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“…There are thermostable α-amylases from plants, fungi, animals and microbes [ 44 ]. Several of these enzymes come from hyperthermophilic archaea belonging to the genera Pyrococcus [ 45 , 46 ], Thermococcus [ 47 – 49 ], Desulfurococcus [ 50 ] , Staphylothermus [ 50 ], Methanococcus [ 51 ], and Sulfolobus [ 52 ]. In addition, there are also α-amylases from haloalkaliphilic archaea belonging to the genera Haloarcula [ 53 – 55 ], Halorubrum [ 56 ], Haloferax [ 57 ], and Natronococcus [ 58 ] (see Table 5 ).…”

Section: Glycosyl Hydrolases (Ec 321x)mentioning

confidence: 99%

Biotechnological applications of archaeal enzymes from extreme environments

Cabrera

Blamey

2018

Biol Res

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To date, many industrial processes are performed using chemical compounds, which are harmful to nature. An alternative to overcome this problem is biocatalysis, which uses whole cells or enzymes to carry out chemical reactions in an environmentally friendly manner. Enzymes can be used as biocatalyst in food and feed, pharmaceutical, textile, detergent and beverage industries, among others. Since industrial processes require harsh reaction conditions to be performed, these enzymes must possess several characteristics that make them suitable for this purpose. Currently the best option is to use enzymes from extremophilic microorganisms, particularly archaea because of their special characteristics, such as stability to elevated temperatures, extremes of pH, organic solvents, and high ionic strength. Extremozymes, are being used in biotechnological industry and improved through modern technologies, such as protein engineering for best performance. Despite the wide distribution of archaea, exist only few reports about these microorganisms isolated from Antarctica and very little is known about thermophilic or hyperthermophilic archaeal enzymes particularly from Antarctica. This review summarizes current knowledge of archaeal enzymes with biotechnological applications, including two extremozymes from Antarctic archaea with potential industrial use, which are being studied in our laboratory. Both enzymes have been discovered through conventional screening and genome sequencing, respectively.

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Section: Glycosyl Hydrolases (Ec 321x)mentioning

confidence: 99%

Biotechnological applications of archaeal enzymes from extreme environments

Cabrera

Blamey

2018

Biol Res

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“…is widely used for thermostable α-amylase production to meet industrial needs [9,10]. In addition, the most thermoactive α-amylases from hyperthermophilic archaea have attracted increasing attention and have been characterized from Pyrococcus woesei , P. furiosus , Thermococcus profundus , and T. hydrothermalis [11-14]. Some general strategies to increase the thermal stability of these enzymes have also been proposed and used for directed evolution, such as change of the secondary structure strengthening the hydrophobic interactions in intermolecular contacts, introduction of hydrogen bonds and salt bridges, and increase of the hydrophobicity of the protein surface [15,16].…”

Section: Introductionmentioning

confidence: 99%

Close relationship of a novel Flavobacteriaceaeα-amylase with archaeal α-amylases and good potentials for industrial applications

Cheng

et al. 2014

Biotechnol Biofuels

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BackgroundBioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process.ResultsA novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.ConclusionsThe improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

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“…Mesophilic FSA from bacteria is evolutionary closely related to thermophilic archaeal PWA, but displays a sharply decreased thermostability, thus providing an ideal target for revealing strategies for the adaption of this mesophilic protein to a mesophilic environment. As an important signal for lateral gene transfer from PWA to FSA, the uncommon (Ca, Zn) two-metal center only exists in a few GH13_7 α-amylases, including PWA and those of some Thermococcus species 27 , 29 , 31 , 32 . By site-directed mutagenesis, both Ca 2+ - and Zn 2+ -binding sites were found to be important for the thermostability of PWA 27 , 28 .…”

Section: Discussionmentioning

confidence: 99%

Functional and cooperative stabilization of a two-metal (Ca, Zn) center in α-amylase derived from Flavobacteriaceae species

Yin

Zhou

Nie

et al. 2017

Sci Rep

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Mesophilic α-amylase from Flavobacteriaceae (FSA) is evolutionary closely related to thermophilic archaeal Pyrococcus furiosus α-amylase (PWA), but lacks the high thermostability, despite the conservation of most residues involved in the two-metal (Ca, Zn) binding center of PWA. In this study, a disulfide bond was introduced near the two-metal binding center of FSA (designated mutant EH-CC) and this modification resulted in a slight improvement in thermostability. As expected, E204G mutations in FSA and EH-CC led to the recovery of Ca2+-binding site. Interestingly, both Ca2+- and Zn2+-dependent thermostability were significantly enhanced; 153.1% or 50.8% activities was retained after a 30-min incubation period at 50 °C, in the presence of Ca2+ or Zn2+. The C214S mutation, which affects Zn2+-binding, also remarkably enhanced Zn2+- and Ca2+- dependent thermostability, indicating that Ca2+- and Zn2+-binding sites function cooperatively to maintain protein stability. Furthermore, an isothermal titration calorimetry (ITC) analysis revealed a novel Zn2+-binding site in mutant EH-CC-E204G. This metal ion cooperation provides a possible method for the generation of α-amylases with desired thermal properties by in silico rational design and systems engineering, to generate a Zn2+-binding site adjacent to the conserved Ca2+-binding site.

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Cloning and expression of an Î±-amylase encoding gene from the hyperthermophilic archaebacteriumThermococcus hydrothermalisand biochemical characterisation of the recombinant enzyme

Cited by 17 publications

References 21 publications

Biotechnological applications of archaeal enzymes from extreme environments

Biotechnological applications of archaeal enzymes from extreme environments

Close relationship of a novel Flavobacteriaceaeα-amylase with archaeal α-amylases and good potentials for industrial applications

Functional and cooperative stabilization of a two-metal (Ca, Zn) center in α-amylase derived from Flavobacteriaceae species

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