scholarly journals The Thermostable Enzyme Genes of the dTDP-L-Rhamnose Synthesis Pathway (rmlBCD) from a Thermophilic Archaeon

10.5772/29196 ◽  
2012 ◽  
Author(s):  
Maki Teramoto ◽  
Zilian Zhang ◽  
Motohiro Shizuma ◽  
Takashi Kawasaki ◽  
Yutaka Kawarabayasi ◽  
...  
Keyword(s):  
Thermostable Enzyme ◽  
Thermophilic Archaeon

scholarly journals Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Yuki Honda ◽  
Qian Zang ◽  
Yasuhiro Shimizu ◽  
Mohammad Dadashipour ◽  
Zilian Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Thermostable Enzyme ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Content Type ◽  
Mutant Proteins ◽  
Sulfolobus Tokodaii ◽  
Site Saturation ◽  
Thermophilic Archaeon

ABSTRACT The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii. Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.

scholarly journals A tungsten-containing active formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei

1992 ◽  
Vol 207 (2) ◽  
pp. 559-565 ◽  
Author(s):  
Ruth A. SCHMITZ ◽  
Monika RICHTER ◽  
Dietmat LINDER ◽  
Rudolf K. THAUER
Keyword(s):  
Thermophilic Archaeon

scholarly journals A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity

2000 ◽  
Vol 182 (22) ◽  
pp. 6424-6433 ◽  
Author(s):  
Masaru Nakatani ◽  
Satoshi Ezaki ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Biochemical Study ◽  
Protein Product ◽  
Optimum Concentration ◽  
Dna Ligase ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon ◽  
Dna Ligases ◽  
Cofactor Specificity ◽  
Thermophilic Archaeon ◽  
Optimum Ph

ABSTRACT A gene encoding DNA ligase (ligTk ) from a hyperthermophilic archaeon, Thermococcus kodakaraensisKOD1, has been cloned and sequenced, and its protein product has been characterized. ligTk consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig Tk was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig Tk was closely related to the ATP-dependent DNA ligase fromMethanobacterium thermoautotrophicum ΔH, a moderate thermophilic archaeon, along with putative DNA ligases fromEuryarchaeota and Crenarchaeota. We expressedligTk in Escherichia coli and purified the recombinant protein. Recombinant Lig Tk was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg2+. The optimum pH of Lig Tk was 8.0, the optimum concentration of Mg2+, which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K+ was 10 to 30 mM. Lig Tk did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100°C. Unexpectedly, Lig Tk displayed a relatively small, but significant, DNA ligase activity when NAD+ was added as the cofactor. Treatment of NAD+ with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD+ solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig Tk with NAD+. This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.

scholarly journals High stability of trehalose/maltose binding protein fromThermococcus litoralismakes it a good candidate as a sensitive element in biosensor systems for sugar control

2010 ◽  
Vol 24 (3-4) ◽  
pp. 349-353 ◽  
Author(s):  
Olga I. Povarova ◽  
Olga V. Stepanenko ◽  
Anna I. Sulatskaya ◽  
Irina M. Kuznetsova ◽  
Konstantin K. Turoverov ◽  
...  
Keyword(s):  
Free Energy ◽  
Sensitive Element ◽  
High Stability ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Thermococcus Litoralis ◽  
Thermophilic Archaeon ◽  
Unfolded State ◽  
The Stability ◽  
Innate Ability

Fluorescence and circular dichroism in far-UV region were used to study the stability of trehalose/maltose binding protein (TMBP) from hyper thermophilic archaeonThermococcus litoralisand its complex with glucose (TMBP/Glc). The evaluation of difference between free energy of native and unfolded state for TMBP and TMBP/Glc showed that both of them are several times higher than that of proteins from mesophilic organisms. Due to the high stability and innate ability to bind glucose this protein is a good candidate as a sensitive element in biosensor systems for sugar control.

Production of Highly Thermostable Laccase from Penicillium spinulosum for Enhanced Delignification of Untreated Wheat Bran

2021 ◽  
Vol 10 ◽  
Author(s):  
Folasade M. Olajuyigbe ◽  
Ademola K. Oduwole ◽  
Cornelius O. Fatokun
Keyword(s):  
Physicochemical Properties ◽  
Wheat Bran ◽  
Plant Cell Wall ◽  
Palm Kernel ◽  
Residual Activity ◽  
Thermostable Enzyme ◽  
Biofuel Production ◽  
Laccase Production ◽  
Trichoderma Species ◽  
High Thermostability

Background: Lignin confers rigidity on plant cell wall and poses a challenge to hydrolysis of cellulose, which makes production of biofuels from lignocellulose an overwhelming problem. This prompts continuous search for novel ligninolytic enzymes, especially, laccases for delignification of lignocellulose for improved saccharification of biomass. Objective: This study reports production, physicochemical properties and delignification efficiency of laccases from Penicillium and Trichoderma species on untreated wheat bran. Methods: Fungal laccases were produced using different agroresidues (wheat bran, coconut shell and palm kernel shell) as substrates in submerged fermentation. Best substrate for laccase production was determined. Physicochemical properties of crude enzymes and delignification efficiency of the laccases were determined on untreated wheat bran using pure laccase as control. Results: Wheat bran supported maximum laccase production from fungi under study. Highest laccase yield of 22.5 U/mL was obtained from P. spinulosum. Laccase from P. spinulosum was optimally active at pH 7.0 and 50 °C and exhibited remarkable high thermostability with 61.6% residual activity at 90 °C after 2 h incubation. The activity of the thermostable enzyme was enhanced in the presence of Cu2+. Biodelignification efficiency of cell-free extract from P. spinulosum, T. koningii, and P. restrictum on wheat bran were 95%, 81.5% and 63.5%, respectively. Surprisingly, a much lower delignification efficiency of 33.42% was obtained with commercial laccase from Trametes versicolor. Conclusion: The high thermostability and striking delignification efficiency of laccase from P. spinulosum make the enzyme a good bioresource for biodelignification of untreated lignocellulose for biofuel production.

scholarly journals A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0125325 ◽  
Author(s):  
Sangmin Lee ◽  
Baolei Jia ◽  
Jinliang Liu ◽  
Bang Phuong Pham ◽  
Jae Myeong Kwak ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Thermophilic Archaeon

scholarly journals Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

1998 ◽  
Vol 180 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Masahiro Furutani ◽  
Toshii Iida ◽  
Shigeyuki Yamano ◽  
Kei Kamino ◽  
Tadashi Maruyama
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ppiase Activity ◽  
Methanococcus Thermolithotrophicus ◽  
Fk506 Binding Protein ◽  
Thermophilic Archaeon

ABSTRACT A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.

scholarly journals Thermophilic Lifestyle for an Uncultured Archaeon from Hydrothermal Vents: Evidence from Environmental Genomics

2006 ◽  
Vol 72 (3) ◽  
pp. 2268-2271 ◽  
Author(s):  
Hélène Moussard ◽  
Ghislaine Henneke ◽  
David Moreira ◽  
Vincent Jouffe ◽  
Purificacion López-García ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Dna Polymerase ◽  
Deep Sea ◽  
Hydrothermal Vents ◽  
Way Of Life ◽  
Thermoplasma Acidophilum ◽  
Environmental Genomics ◽  
Optimal Activity ◽  
Thermophilic Archaeon ◽  
Uncultured Archaea

ABSTRACT We present a comparative analysis of two genome fragments isolated from a diverse and widely distributed group of uncultured euryarchaea from deep-sea hydrothermal vents. The optimal activity and thermostability of a DNA polymerase predicted in one fragment were close to that of the thermophilic archaeon Thermoplasma acidophilum, providing evidence for a thermophilic way of life of this group of uncultured archaea.

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