Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli.

Laderman, Kenneth A.; Asada, Kazumi; Uemori, Takashi; Mukai, Hideyuki; Taguchi, Yoshitaka; Kato, Ikuo; Anfinsen, Christian B.

doi:10.1016/s0021-9258(20)80539-0

Cited by 118 publications

(7 citation statements)

References 18 publications

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“…Most of these enzymes come from thermophilic archaea, like Pyrococcus furiosus , Thermococcus kodakarensis and Sulfolobus solfataricus , while the remainder come from thermophilic bacteria [ 7 - 9 ]. P. furiosus α-amylase is a hyperthermophilic enzyme that exhibits optimal activity at about 100°C, where it has a half-life of more than 12 h [ 10 , 11 ]. In addition to its excellent thermostability, P. furiosus α-amylase exhibits optimal activity at a pH of about 5.6, with good stability and no less than 80% of optimal activity at pH values from 4.5 to 6.5.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization

Zhang

Tan

Yao

et al. 2021

J. Microbiol. Biotechnol.

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Alpha-amylase (E.C. 3.2.1.1) is an enzyme that catalyzes the hydrolysis of α-1,4 linkages in starch and related carbohydrates and has been widely applied in starch processing, baking, brewing and the de-sizing of textiles [1][2][3]. In industrial starch processing, α-amylases are used in the liquefaction step to hydrolyze starch into glucose oligomers. The ideal temperature and pH for this step are 105 o C and pH 4.5, respectively. Since 1980, Bacillus licheniformis α-amylase has been the main enzyme used in starch liquefaction. This enzyme exhibits optimal activity at 90 o C and pH 6.0, which can only partly satisfy the starch liquefaction requirement [4][5][6]. Hyperthermophilic enzymes exhibit optimal activity at temperatures higher than 90 o C. Most of these enzymes come from thermophilic archaea, like Pyrococcus furiosus, Thermococcus kodakarensis and Sulfolobus solfataricus, while the remainder come from thermophilic bacteria [7][8][9]. P. furiosus α-amylase is a hyperthermophilic enzyme that exhibits optimal activity at about 100 o C, where it has a half-life of more than 12 h [10,11]. In addition to its excellent thermostability, P. furiosus α-amylase exhibits optimal activity at a pH of about 5.6, with good stability and no less than 80% of optimal activity at pH values from 4.5 to 6.5. These characteristics make P. furiosus α-amylase potentially suitable for industrial applications.P. furiosus α-amylase has been known and characterized since 1990, but the relatively low level of heterologous expression has limited its industrial application [2]. When produced in Escherichia coli, P. furiosus α-amylase is Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100°C), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90 o C for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3-and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. f...

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Section: Introductionmentioning

confidence: 99%

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization

Zhang

Tan

Yao

et al. 2021

J. Microbiol. Biotechnol.

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“…It grows optimally at 98-100 °C (16) and can utilize a number of carbohydrates, including starch, pullulan, and maltose (17), as well as cellobiose (18) and laminarin (19). To assimilate these compounds, P. furiosus must necessarily produce several glycosyl hydrolases, including two amylases (20)(21)(22), an amylopullulanase (17,23), an R-glucosidase (24), a β-glucosidase (18,25,26), a β-mannosidase (26), and a laminarinase (19). The exact mechanism of action has been elucidated neither for these enzymes from P. furiosus nor for any glycosyl hydrolase from a thermophilic organism nor for any glycosyl hydrolase from an archaeon.…”

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confidence: 99%

The Family 1 β-Glucosidases from Pyrococcus furiosus and Agrobacterium faecalis Share a Common Catalytic Mechanism

Bauer

Kelly

1998

Biochemistry

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Comparisons of catalytic mechanisms have not previously been performed for homologous enzymes from hyperthermophilic and mesophilic sources. Here, the beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was recombinantly produced in Escherichia coli and shown to have biophyscial and biochemical properties identical to those of the wild-type enzyme. Moreover, the recombinant enzyme was subjected to a detailed kinetic investigation at 95 degreesC to compare its catalytic mechanism to that determined at 37 degreesC for the beta-glucosidase (abg) from the mesophilic bacterium, Agrobacterium faecalis [Kempton, J., and Withers, S. G. (1992) Biochemistry 31, 9961]. These enzymes have amino acid sequences that are 33% identical and have been classified as family 1 glycosyl hydrolases on the basis of amino acid sequence similarities. Both enzymes have similar broad specificities for both sugar and aglycone moieties and exhibit nearly identical pH dependences for their kinetic parameters with several different substrates. Bronsted plots were constructed for bgl at several temperatures using a series of aryl glucoside substrates. These plots were concave downward at all temperatures, indicating that bgl utilized a two-step mechanism similar to that of abg and that the rate-limiting step in this mechanism did not change with temperature for any given aryl glucoside. The Bronsted coefficient for bgl at 95 degreesC (beta1g = -0.7) was identical to that for abg at 37 degreesC and implies that these enzymes utilize nearly identical transition states, at least in regard to charge accumulation on the departing glycosidic oxygen. In addition, a high correlation coefficient (rho = 0.97) for the linear free energy relationship between these two enzymes and similar inhibition constants for these two enzymes with several ground state and transition state analogue inhibitors further indicate that these enzymes stabilize similar transition states. The mechanistic similarities between these two enzymes are noteworthy in light of the large difference in their temperature optima. This suggests that, in the presumed evolution that occurred between the hyperthermophilic archaeal enzyme and the mesophilic bacterial enzyme, structural modifications must have been selected which maintained the integrity of the active site structure and, therefore, the specificity of transition state interactions, while adapting the overall protein structure to permit function at the appropriate temperature.

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“…The family GH57 was established in 1996 by the second published update of the GH classification [6]. The main reason for creating this family was two presumed α-amylases -one from the bacterium Dictyoglomus thermophilum [83] and the other from the archaeon Pyrococcus furiosus [84]. Their amino acid sequences were obviously different from those already classified in family GH13 [30], although efforts to reveal sequence features joining GH13 and GH57 persisted at the time, mainly owing to the lack of structural data [85].…”

Section: The Main α-Amylase Family Gh13mentioning

confidence: 99%

“…Currently (November 2021), family GH57 has almost 4,000 sequences from prokaryotes, roughly divided 3:1 between Bacteria and Archaea [1]. However, it is worth remarking that the two founding family members are in fact 4-α-glucanotransferases [93,94], but were originally supposed to be α-amylases from D. thermophilum [83] and P. furiosus [84]. Moreover, yet another member that may be crucial for naming family GH57 as "α-amylase" family is the α-amylase from Methanocaldococcus jannaschii [85], which is an amylopullulanase and not a strict α-amylase as it showed > 80% activity on pullulan compared to on soluble starch [95].…”

Section: The Main α-Amylase Family Gh13mentioning

confidence: 99%

How many α-amylase GH families are there in the CAZy database?

Janeček

Svensson

2022

Amylase

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The CAZy database is a web-server for sequence-based classification of carbohydrate-active enzymes that has become the worldwide and indispensable tool for scientists engaged in this research field. It was originally created in 1991 as a classification of glycoside hydrolases (GH) and currently, this section of CAZy represents its largest part counting 172 GH families. The present Opinion paper is devoted to the specificity of α-amylase (EC 3.2.1.1) and its occurrence in the CAZy database. Among the 172 defined GH families, four, i.e. GH13, GH57, GH119 and GH126, may be considered as the α-amylase GH families. This view reflects a historical background and traditions widely accepted during the previous decades with respect to the chronology of creating the individual GH families. It obeys the phenomenon that some amylolytic enzymes, which were used to create the individual GH families and were originally known as α-amylases, according to current knowledge from later, more detailed characterization, need not necessarily represent genuine α-amylases. Our Opinion paper was therefore written in an effort to invite the scientific community to think about that with a mind open to changes and to consider the seemingly unambiguous question in the title as one that may not have a simple answer.

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Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli.

Cited by 118 publications

References 18 publications

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization

The Family 1 β-Glucosidases from Pyrococcus furiosus and Agrobacterium faecalis Share a Common Catalytic Mechanism

How many α-amylase GH families are there in the CAZy database?

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