scholarly journals Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album

2022 ◽  
Vol 25 (8) ◽  
pp. 882-888
Author(s):  
A. B. Beklemishev ◽  
M. B. Pykhtina ◽  
Ya. M. Kulikov ◽  
T. N. Goryachkovskaya ◽  
D. V. Bochkov ◽  
...  
Keyword(s):  
Culture Medium ◽  
Methylotrophic Yeast ◽  
Yeast Cells ◽  
Proteinase K ◽  
Subsequent Stage ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
E Coli ◽  
Komagataella Phaffii ◽  
Biological Methods

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

scholarly journals Adhesion ofE. coliBacteria Cells to Prosthodontic Alloys Surfaces Modified by TiO2Sol-Gel Coatings

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Katarzyna Banaszek ◽  
Witold Szymanski ◽  
Bożena Pietrzyk ◽  
Leszek Klimek
Keyword(s):  
Titanium Dioxide ◽  
Bacterial Adhesion ◽  
Culture Medium ◽  
Bacterial Culture ◽  
Bare Metal ◽  
Bacterial Cells ◽  
E Coli ◽  
Modified Surfaces ◽  
Bare Substrate ◽  
Scanning Electron

The evaluation of the degree of bacteriaE. coliadhesion to modified surfaces of the chosen prosthodontic alloys was presented. The study was carried out on Co-Cr (Wironit), Ni-Cr (Fantocer), and Fe-Cr-Ni (Magnum AN) alloys. Bare substrate as a control and titanium dioxide coated samples were used. The samples were placed for 24 hours in bacterial culture medium. After incubation period, a number of bacterial cells were evaluated by scanning electron microscope. The study revealed that modification of the alloy surfaces by titanium dioxide coating significantly decreases the amount of bacteria adhering to the surfaces and that additionally bare metal alloy substrates have a different degree of susceptibility to bacterial adhesion.

scholarly journals Extracellular Production, Characterization, and Engineering of a Polyextremotolerant Subtilisin-Like Protease From Feather-Degrading Thermoactinomyces vulgaris Strain CDF

2020 ◽  
Vol 11 ◽  
Author(s):  
Yidi Ding ◽  
Yong Yang ◽  
Yuxia Ren ◽  
Jingying Xia ◽  
Feng Liu ◽  
...  
Keyword(s):  
Proteinase K ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
E Coli ◽  
Laundry Detergents ◽  
Subtilisin Carlsberg ◽  
Nacl Concentration ◽  
Direct Substitution ◽  
Thermoactinomyces Vulgaris ◽  
Commercial Laundry

Here, the gene encoding a subtilisin-like protease (protease Als) was cloned from Thermoactinomyces vulgaris strain CDF and expressed in Escherichia coli. The recombinant enzyme was released into the culture medium of E. coli as a mature form (mAls). Purified mAls displayed optimal activity at 60–70°C and pH 10.0 using azo-casein as the substrate, and showed a half-life of 13.8 h at 70°C. Moreover, the activity of thermostable mAls was comparable to or higher than those of mesophilic subtilisin Carlsberg and proteinase K at low temperatures (10–30°C). Protease Als was also stable in several organic solvents and showed high compatibility with commercial laundry detergents. Notably, mAls exhibited approximately 100% of its activity at 3 M NaCl, and showed enhanced thermostability with the increase of NaCl concentration up to 3 M. Protease Als possesses an excess of solvent-accessible acidic amino acid residues, which may account for the high halotolerance of the enzyme. Compared with homologous protease C2 from the same strain, protease Als exhibits substantially lower activity toward insoluble keratin substrates but efficiently hydrolyzes soluble keratin released from chicken feathers. Additionally, direct substitution of the substrate-binding site of protease Als with that of protease C2 improves its activity against insoluble keratin substrates. By virtue of its polyextremotolerant attribute and kerationolytic capacity, protease Als may find broad applications in various industries such as laundry detergents, food processing, non-aqueous biocatalysis, and feather processing.

scholarly journals Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the EnteroaggregativeEscherichia coliStrain 042

mSystems ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
A. Prieto ◽  
M. Bernabeu ◽  
S. Aznar ◽  
S. Ruiz-Cruz ◽  
A. Bravo ◽  
...  
Keyword(s):  
Protein A ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Specific Expression ◽  
Protein Targets ◽  
Global Regulators ◽  
E Coli ◽  
The Family ◽  
Regulatory Properties

ABSTRACTBacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the familyEnterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. InEscherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregativeE. colistrain 042 carries, in addition to thehnsandstpAgenes, a third gene encoding anhnsparalogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within theEnterobacteriaceae.IMPORTANCEGlobal regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregativeE. colistrain 042 carries a new hitherto uncharacterized copy of thehnsgene. We decided to investigate why this pathogenicE. colistrain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

scholarly journals Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court
Keyword(s):  
Dna Assembly ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Engineering Technology ◽  
E Coli ◽  
Linear Dna ◽  
Recombination System ◽  
Genetic Engineering Technology

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

scholarly journals Involvement of a Sialic Acid-Binding Lectin with Hemagglutination and Hydrophobicity of Flavobacterium psychrophilum

2003 ◽  
Vol 69 (9) ◽  
pp. 5275-5280 ◽  
Author(s):  
Jeannette Dan Møller ◽  
Jens Laurits Larsen ◽  
Lone Madsen ◽  
Inger Dalsgaard
Keyword(s):  
Rainbow Trout ◽  
Sialic Acid ◽  
Periodate Oxidation ◽  
Hydrophobic Interaction Chromatography ◽  
Yeast Cells ◽  
Proteinase K ◽  
Bacterial Cells ◽  
Flavobacterium Psychrophilum ◽  
Proteinase K Treatment ◽  
Highly Hydrophobic

ABSTRACT Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and FpT exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5°C, compared to that at 15°C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65°C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.

scholarly journals Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence

2003 ◽  
Vol 69 (11) ◽  
pp. 6393-6398 ◽  
Author(s):  
N. A. Romanova ◽  
L. Y. Brovko ◽  
L. Moore ◽  
E. Pometun ◽  
A. P. Savitsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Yeast Cells ◽  
Bacterial Cells ◽  
E Coli ◽  
Intracellular Atp ◽  
Microdilution Method ◽  
Wide Range ◽  
Atp Bioluminescence ◽  
Time Courses

ABSTRACT Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

scholarly journals A Comparative Study On Self-Healing Methods of Concretes By Sporosarcina Pasteurii Bacteria

2021 ◽  
Author(s):  
Mohammad Mirshahmohammad ◽  
Hamid Rahmani ◽  
Mahdi Maleki-Kakelar ◽  
Abbas Bahari
Keyword(s):  
Culture Medium ◽  
Healing Process ◽  
Calcium Nitrate ◽  
Healing Time ◽  
Concrete Mixture ◽  
Bacterial Cells ◽  
Self Healing ◽  
Calcium Carbonate Precipitation ◽  
Sporosarcina Pasteurii ◽  
Biological Methods

Abstract Biological methods (adding bacteria to the concrete mixtures) among the most recently investigated procedures increase the durability of concrete and repair concrete cracks. In the present study, different biological methods were used to heal the cracks of concrete and the most suitable method was subsequently introduced. For this purpose, the culture medium and bacterial nutrient inside the concrete mixes and curing solution were separately studied. The effect of air-entrained agent and various sources of calcium salts as the bacterial nutrient on the healing process was also studied. The results showed that the use of bacterial nutrient inside the concrete mixes has an affirmative impact on the mechanical properties and self-healing characteristics of concretes. With the simultaneous use of Sporosarcina pasteurii bacteria and calcium nitrate-urea or calcium chloride-urea as a bacterial nutrient in the concrete mixture, the 28 days compressive strength of concrete increases by 23.4% and 7.5%, respectively, which is due to calcium carbonate precipitation. The use of bacterial cells, nutrients, and culture in the concrete mixture provided the ability to heal wide cracks where the healing time is significantly reduced. On the other hand, separation of the bacterial culture medium slightly reduced the self-healing performance of concrete.

scholarly journals The Global Regulatory hns Gene Negatively Affects Adhesion to Solid Surfaces by Anaerobically Grown Escherichia coli by Modulating Expression of Flagellar Genes and Lipopolysaccharide Production

2002 ◽  
Vol 184 (6) ◽  
pp. 1522-1529 ◽  
Author(s):  
Paolo Landini ◽  
Alexander J. B. Zehnder
Keyword(s):  
Escherichia Coli ◽  
Sharp Decrease ◽  
Luria Broth ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Aerobic Conditions ◽  
E Coli ◽  
Hydrophilic Surfaces ◽  
K 12 ◽  
Initial Binding

ABSTRACT The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation. In this report we show that stationary-phase cultures of Escherichia coli W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28°C in fully aerobic conditions. In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates. We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the fliC gene, encoding the major flagellar subunit, increases under oxygen-limited conditions. Inactivation of the global regulatory hns gene counteracts increased production of LPS and flagella in response to anoxia and allows E. coli W3100 to attach to sand columns even when it is grown under oxygen-limited conditions. We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of E. coli W3100 to adhere to hydrophilic surfaces. Indeed, overexpression of the fliC gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions. Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in E. coli.

Exogenous Histidine as a Co-factor which Exacerbates the Toxicity of Hydrogen Peroxide in Cultured Mammalian and Bacterial Cells

1991 ◽  
Vol 19 (1) ◽  
pp. 68-70
Author(s):  
Giorgio Brandi ◽  
Piero Sestili ◽  
Andrea Guidarelli ◽  
Giuditta Fiorella Schiavano ◽  
Amedeo Albano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Chinese Hamster ◽  
Bacterial Cells ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Casamino Acids

The killing of Escherichia coli cells by H2O2 is higher when exposure to the oxidant is performed in a complete culture medium, as compared to saline. Whereas MgSO4, CaCl2, thiamine or glucose, added separately or in combination with the saline, had no effect on the cytotoxic response to H2O2, the cytotoxicity appeared highly dependent upon the presence of the casamino acids in the incubation medium. One of these amino acids, histidine, was found to greatly augment the toxicity of H2O2 in E. coli. This effect of histidine was also observed in mammalian cells. In fact, both the cytoxicity and the DNA damage produced by H2O2 in Chinese hamster ovary (CHO) cells were significantly increased by this amino acid.

scholarly journals Cloning, Overexpression, and Mutagenesis of theSporobolomyces salmonicolor AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (S)-4-Chloro-3-Hydroxybutanoate

1999 ◽  
Vol 65 (12) ◽  
pp. 5207-5211 ◽  
Author(s):  
Keiko Kita ◽  
Takanobu Fukura ◽  
Koh-Ichi Nakase ◽  
Kenji Okamoto ◽  
Hideshi Yanase ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Inhibition ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Binding Motif ◽  
Aldehyde Reductase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
E Coli

ABSTRACT We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolorAKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced inEscherichia coli about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed inE. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G19-X-X-G22-X-X-A25, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G19→A and G22→A mutant enzymes by 4-COBE did not occur. The A25→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

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