A novel phytase from Citrobacter gillenii: characterization and expression in Pichia pastoris (Komagataella pastoris)

2020 ◽  
Author(s):  
Artur A Tkachenko ◽  
Anna N Kalinina ◽  
Larisa N Borshchevskaya ◽  
Sergey P Sineoky ◽  
Tatiana L Gordeeva
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Gene Encoding ◽  
Maximum Reaction Rate ◽  
Maximum Reaction ◽  
Wide Ph Range ◽  
Recombinant Phytase ◽  
Ph Range

Abstract The phyCg gene encoding a new phytase from C. gillenii was optimized, synthesized, cloned, and expressed in Pichia pastoris. Analysis of the amino acid sequence of the enzyme showed that it belongs to the histidine acid phosphatase family. The amino acid sequence of the PhyCg phytase has the highest homology (73.49%) with a phytase sequence from Citrobacter braakii. The main characteristics for the purified recombinant phytase were established. The optimum pH and temperature were 4.5 and 50°C, respectively. The specific activity of the enzyme was 1577 U/mg. The Michaelis constant (Km) and the maximum reaction rate (Vmax) for sodium phytate were 0,185 mM and 2185 U/mg, respectively. The enzyme showed the pH and trypsin stability and had a high activity over a wide pH range.

New Recombinant Phytase from Kosakonia sacchari: characteristic and biotechnological potential

2019 ◽  
Vol 35 (4) ◽  
pp. 33-41
Author(s):  
L.N. Borshchevskaya ◽  
A.N. Kalinina ◽  
N.V. Bulushova ◽  
S.P. Syneoky ◽  
S.P. Voronin ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Ministry Of Education ◽  
Maximum Reaction Rate ◽  
Biotechnological Potential ◽  
Resource Center ◽  
Codon Composition ◽  
Maximum Reaction ◽  
Wide Range ◽  
Recombinant Phytase

A DNA sequence from Kosakonia sacchari that according to automated computer analysis is believed to correspond to a gene for histidine acid phytase has been selected from the GenBank database. The sequence was optimized for codon composition, synthesized, cloned and expressed in Pichia pastoris. Main characteristics of the purified recombinant enzyme were determined. It was established that the values of pH=4.5 and temperature of 50 °C are optimal for the phytase functioning. The values of specific activity, Michaelis constant (Km) and maximum reaction rate (Vmax) with phytate as a substrate were 1470 U/mg, 193 uM and 2167 umol/(min ∙ mg), respectively. It was shown that the enzyme was characterized by a wide range of working pH. Therefore, the properties of a new recombinant phytase allow us to consider it as a high-potential enzyme for agrobiotechnology. histidine acid phosphatases, Kosakonia sacchari phytase, Pichia pastoris The work was financially supported by the Ministry of Education and Science of Russian Federation (Unique Project Identifier RFMEFI57917X0145) and was carried out using the Multipurpose Scientific Installation of National Bio-resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

Expression of Aspergillus aculeatus β-mannanase in Pichia pastoris Yeast and Analysis of Industrially Important Properties of Enzyme

2019 ◽  
Vol 35 (1) ◽  
pp. 38-44
Author(s):  
M.N. Lazareva ◽  
E.I. Semenko ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
High Specific Activity ◽  
Yeast Cells ◽  
Aspergillus Aculeatus ◽  
Gene Encoding ◽  
Pichia Pastoris Yeast ◽  
Efficient Expression ◽  
Ph Range ◽  
First Time

β-Mannanases are enzymes for the industrial application and they can be used, in particular, in the feed industry. The most important requirements for feed enzymes are broad pH range, thermal stability and high specific activity. The efficient expression of the man1 gene encoding Aspergillus aculeatus β-1,4-mannanases in Pichia pastoris yeast cells has been obtained for the first time. The industrially valuable properties of the enzyme were confirmed. The obtained data indicate that the man1 gene from A. aculeatus is potentially useful for the construction of industrial mannanase producers on the basis of the Pichia pastoris yeast. recombinant β-mannanase, Pichia pastoris, Aspergillus aculeatus, overexpression. The work was financially supported by State project №595-00004-18 PR and used the help of the National Bioresource Center - Russian National Collection of Industrial Microorganisms NRC «Kurchatov Institute» - GosNIIgenetika (Moscow, Russia).

scholarly journals Production Optimization of an Active β-Galactosidase of Bifidobacterium animalis in Heterologous Expression Systems

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Xinxin Xu ◽  
Xiaohu Fan ◽  
Chao Fan ◽  
Xing Qin ◽  
Bo Liu ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Culture Medium ◽  
Specific Activity ◽  
Food Additives ◽  
Production Optimization ◽  
E Coli ◽  
Bifidobacterium Animalis ◽  
Wide Ph Range ◽  
Heterologous Expression Systems ◽  
Ph Range

β-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0–8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.

scholarly journals Genetic and Biochemical Characterization of a Highly Thermostable α-l-Arabinofuranosidase fromThermobacillus xylanilyticus

2000 ◽  
Vol 66 (4) ◽  
pp. 1734-1736 ◽  
Author(s):  
Takoua Debeche ◽  
Nicola Cummings ◽  
Ian Connerton ◽  
Philippe Debeire ◽  
Michael J. O'Donohue
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Classification System ◽  
Biochemical Characterization ◽  
Glycosyl Hydrolase ◽  
Gene Encoding ◽  
Ph Range ◽  
Sequence Similarities

ABSTRACT The gene encoding an α-l-arabinofuranosidase fromThermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90°C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56,071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.

Cloning of a Cellobiohydrolase Gene (cbh1) from Aspergillus niger and Heterogenous Expression in Pichia pastoris

2011 ◽  
Vol 347-353 ◽  
pp. 2443-2447
Author(s):  
Guo Qing Li ◽  
Chang Sheng Chai ◽  
Song Fan ◽  
Lin Guo Zhao
Keyword(s):  
Amino Acid ◽  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Working Condition ◽  
Industrial Applications ◽  
Gene Encoding ◽  
Acid Protein ◽  
Wide Range ◽  
Ph Range ◽  
Amino Acid Protein

A gene encoding a cellobiohydrolase (CBH) was isolated from Aspergillus niger-NL-1 and designated as cbh1. The cbh1 gene contains 1,515 nucleotides with three introns and encodes a 452-amino acid protein with a molecular weight of approximately 60 kDa. The amino acid sequence encoded by cbh1 shows high homology with the sequence of glycoside hydrolase fimily 7. The cellobiohydrolase (cbh1) gene was succussfully expressed in Pichia pastoris KM71H. The recombinant CBHⅠshowed an optimal working condition at 60 °C, pH 4.0 with Kmand Vmaxtoward CMC-Na of 13.81 mM and 0.269 μmol/min, respectively. The enzyme retained more than 80 % of its initial activity after 2 h of incubation at 90 °C and was stable in pH range 1.0～10.0. Because of its moderately stable at high temperature and stability through wide range of pH, this enzyme has potential in various industrial applications.

Identification of the gene encoding 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase in Burkholderia sp. HME13

2020 ◽  
Vol 85 (3) ◽  
pp. 626-629
Author(s):  
Hisashi Muramatsu ◽  
Hiroki Maguchi ◽  
Taisuke Harada ◽  
Takehiro Kashiwagi ◽  
Chul-Sa Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Propionic Acid ◽  
Unknown Function ◽  
Protein Family ◽  
Amino Acid Sequence Analysis ◽  
Gene Encoding

ABSTRACT Here, we report the identification of the gene encoding a novel enzyme, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase, in Burkholderia sp. HME13. The enzyme converts 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid and H2O to 3-(2,5-dioxoimidazolidin-4-yl) propionic acid and H2S. Amino acid sequence analysis of the enzyme indicates that it belongs to the DUF917 protein family, which consists of proteins of unknown function.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.

1988 ◽  
Vol 8 (3) ◽  
pp. 1113-1122 ◽  
Author(s):  
E Czarnecka ◽  
R T Nagao ◽  
J L Key ◽  
W B Gurley
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Stress Protein ◽  
Initiation Site ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Nuclease Mapping

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.

scholarly journals Unique Presence of a Manganese Catalase in a Hyperthermophilic Archaeon, Pyrobaculum calidifontis VA1

2002 ◽  
Vol 184 (12) ◽  
pp. 3305-3312 ◽  
Author(s):  
Taku Amo ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Catalase Activity ◽  
Specific Activity ◽  
Homology Search ◽  
Aerobic Conditions ◽  
Hyperthermophilic Archaeon ◽  
Manganese Catalase ◽  
Catalase Gene ◽  
Pyrobaculum Calidifontis

ABSTRACT We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70°C. The enzyme exhibited a Km value of 170 mM toward H2O2 and a k cat value of 2.9 × 104 s−1·subunit−1 at 25°C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (katPc ). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of katPc revealed that no orthologue genes were present on the archaeal genomes, including those from the “aerobic” (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat Pc can be considered a rare example of a manganese catalase from archaea.

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