scholarly journals Molecular Characterization and Expression of a Phytase Gene from the Thermophilic Fungus Thermomyces lanuginosus

1998 ◽  
Vol 64 (11) ◽  
pp. 4423-4427 ◽  
Author(s):  
Randy M. Berka ◽  
Michael W. Rey ◽  
Kimberly M. Brown ◽  
Tony Byun ◽  
Alan V. Klotz
Keyword(s):  
Aspergillus Niger ◽  
Transcriptional Control ◽  
Specific Activity ◽  
Gene Promoter ◽  
Catalytic Efficiency ◽  
Thermophilic Fungus ◽  
Thermomyces Lanuginosus ◽  
Gene Encoding ◽  
Putative Signal Peptide ◽  
Ph Range

ABSTRACT The phyA gene encoding an extracellular phytase from the thermophilic fungus Thermomyces lanuginosus was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. The phyA gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) with Aspergillus niger phytase. The phyAgene was inserted into an expression vector under transcriptional control of the Fusarium oxysporum trypsin gene promoter and used to transform a Fusarium venenatum recipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 μmol of inorganic phosphate released per min per mg of protein at pH 6 and 37°C. The Thermomyces phytase retained activity at assay temperatures up to 75°C and demonstrated superior catalytic efficiency to any known fungal phytase at 65°C (the temperature optimum). Comparison of this new Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range.

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.

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).

Expression of Gene for β-glucanase from Paenibacillus jamilae Bg1 in Pichia pastoris and Characteristics of Recombinant Enzyme

2019 ◽  
Vol 35 (4) ◽  
pp. 15-23 ◽  
Author(s):  
T.L. Gordeeva ◽  
A.N. Kalinina ◽  
A.V. Serkina ◽  
A.S. Fedorov ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Resource Center ◽  
Putative Signal Peptide ◽  
The Russian Federation ◽  
Paenibacillus Macerans ◽  
Positive Effect

The isolation, heterologous expression and characterization of a new thermostable β-glucanase from Paenibacillus jamilae is described. The bgl26 gene from the P. jamilae Bg1 VKPM B-13093 strain consisting of 714 nucleotides encodes endo-1,3-1,4-β-glucanase (EC 3.2.1.73) containing 213 amino acids and 24 residues of the putative signal peptide in N-end area. The nucleotide sequence of the bgl26 gene and the amino acid sequence of the mature Bgl26 protein have the greatest homology with the sequence of the Paenibacillus macerans endo-l,3-l,4-β-glucanase (82 and 88%, respectively). A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. Purified recombinant enzyme Bgl26 was active towards barley β-glucan. The optimal pH for the enzyme to work was 7,0, and the optimum temperature range was 40-45 °C. The specific activity of β-glucanase was at the level of 6650 U/mg of protein, Km and Vmax were equal to 6.4 ± 0.3 mg/mL and 9450.1 ± 471.2 umol/(min-mg), respectively. The recombinant protein Bgl26 was characterized by high pH and thermal stability, as well as resistance to digestive enzymes. It is also shown that Co2+ ions have a positive effect on the activity of the enzyme. β-glucanase, β-glucan, Paenibacillus jamilae, Pichia pastoris The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) and was carried out using the Multipurpose Scientific Installation of National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

scholarly journals Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

2013 ◽  
Vol 79 (9) ◽  
pp. 3049-3058 ◽  
Author(s):  
Haiquan Yang ◽  
Xinyao Lu ◽  
Long Liu ◽  
Jianghua Li ◽  
Hyun-dong Shin ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Specific Activity ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Content Type ◽  
Resistance To Oxidation ◽  
Catalytic Constant ◽  
N Terminus ◽  
Ph Range

ABSTRACTIn this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) fromAlkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (kcat) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK:kcat/Kmincreased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H2O2, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.

scholarly journals Overexpression of celB Gene Coding for β-Glucosidase from Pyrococcus furiosus Using a Baculovirus Expression Vector System in Silkworm, Bombyx mori

2006 ◽  
Vol 61 (7-8) ◽  
pp. 595-600 ◽  
Author(s):  
Xu’Ai Lin ◽  
Wei Zhang ◽  
Yin Chen ◽  
Bin Yao ◽  
Zhi Fang Zhang
Keyword(s):  
Bombyx Mori ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Divalent Cations ◽  
Gene Promoter ◽  
Protein Quantification ◽  
Vector System ◽  
Glycosyl Hydrolases ◽  
Gene Encoding ◽  
Baculovirus Expression Vector

β-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing β-ᴅ-glucose residues from the end of various oligosaccharides with the release of β-ᴅ-glucose. CelB gene, encoding the thermostable β-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express β-glucosidase in silkworm. The recombinant β-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed β-glucosidase was obtained at pH 5.0 and about 105 °C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant β-glucosidase were similar to the native counterpart. The expressed β-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable β-glucosidase using this system.

scholarly journals Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Samaneh Mosallatpour ◽  
Saeed Aminzadeh ◽  
Mehdi Shamsara ◽  
Reza Hajihosseini
Keyword(s):  
Food Industry ◽  
Tertiary Structure ◽  
Biochemical Characterization ◽  
Arrhenius Plot ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Cysteine Residue ◽  
Enzyme Active Site ◽  
Wide Range ◽  
Ph Range

AbstractL-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

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.

Amylase hyperproduction by deregulated mutants of the thermophilic fungus Thermomyces lanuginosus

2002 ◽  
Vol 29 (2) ◽  
pp. 70-74
Author(s):  
K Rubinder ◽  
BS Chadha ◽  
N Singh ◽  
HS Saini ◽  
S Singh
Keyword(s):  
Thermophilic Fungus ◽  
Thermomyces Lanuginosus

scholarly journals Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

2021 ◽  
Vol 22 (12) ◽  
pp. 6450
Author(s):  
Anita Wiśniewska ◽  
Kamila Wojszko ◽  
Elżbieta Różańska ◽  
Klaudia Lenarczyk ◽  
Karol Kuczerski ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Heterodera Schachtii ◽  
Myb Transcription Factor ◽  
Parasitic Nematodes ◽  
Regulatory Sequences ◽  
Gene Encoding ◽  
Constitutive Overexpression

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

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