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https://www.readbyqxmd.com/read/28428022/cloning-and-identification-of-a-novel-steroid-11%C3%AE-hydroxylase-gene-from-absidia-coerulea
#1
Ruijie Wang, Pengcheng Sui, Xiangjiang Hou, Tong Cao, Longgang Jia, Fuping Lu, Suren Singh, Zhengxiang Wang, Xiaoguang Liu
Steroid 11-hydroxylation by filamentous fungi is a major route for industrial scale production of key intermediates in the synthesis of steroid drugs. Although it is well established that enzymes involved in fungal hydroxylation of steroids are cytochrome P450s (CYP), few fungal steroid hydroxylase genes have been identified. In this study, we identified a novel 11α-hydroxylase gene CYP5311B1 from Absidia coerulea AS3.65 by a combination of transcriptome sequencing, Real-Time qRT-PCR and heterologous expression in Pichia pastoris...
April 17, 2017: Journal of Steroid Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/28425268/efficient-production-of-nanobodies-against-urease-activity-ofhelicobacter-pylori-in-pichia-pastoris
#2
Shahrbanoo Pourasadi, Seyed Latif Mousavi Gargari, Masoumeh Rajabibazl, Shahram Nazarian
BACKGROUND/AIM: Helicobacter pylori is a major health problem. One of the therapeutic approaches is administration of antibody against H. pylori. The methylotrophic Pichia pastoris is a suitable host for expression of recombinant antibody fragments. The aims of this study were the expression and the evaluation of camelid nanobody in the yeast Pichia pastoris. MATERIALS AND METHODS: The camelid-derived heavy-chain antibody (nanobody) against the UreC subunit of urease from H...
April 18, 2017: Turkish Journal of Medical Sciences
https://www.readbyqxmd.com/read/28424672/isolation-and-identification-of-the-indigenous-yeast-population-during-spontaneous-fermentation-of-isabella-vitis-labrusca-l-grape-must
#3
María L Raymond Eder, Cristina Reynoso, Santiago C Lauret, Alberto L Rosa
Grape must harbors a complex community of yeast species responsible for spontaneous alcoholic fermentation. Although there are detailed studies on the microbiota of Vitis vinifera L. grapes, less is known about the diversity and behavior of yeast communities present on fermenting grape must from other species of Vitis. In this work, we used a culture-dependent method to study the identity and dynamics of the indigenous yeast population present during the spontaneous fermentation of Isabella (Vitis labrusca L...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28423625/inhibition-activity-of-a-disulfide-stabilized-diabody-against-basic-fibroblast-growth-factor-in-lung-cancer
#4
Yaxiong Cai, Shuange Yao, Jiangchuan Zhong, Jinxia Zhang, Haowu Jiang, Yanrui Deng, Ning Deng
The over-expression of basic fibroblast growth factor (bFGF) plays a crucial role in the development, invasion and metastasis of lung cancer. Therefore, neutralizing antibodies against bFGF may inhibit the growth of lung cancer. In this study, a Disulfide-stabilized diabody (ds-Diabody) against bFGF was constructed by site-directed mutation and overlap extension PCR (SOE-PCR) at the position of VH44 and VL100 in the scFv. The ds-Diabody was constructed and expressed in Pichia pastoris. We found that the ds-Diabody against bFGF could efficiently suppress the proliferation, migration and invasion of human lung cancer A549 cells in vitro...
March 21, 2017: Oncotarget
https://www.readbyqxmd.com/read/28421142/plackett-burman-design-for-rgilcc1-laccase-activity-enhancement-in-pichia-pastoris-concentrated-enzyme-kinetic-characterization
#5
Edwin D Morales-Álvarez, Claudia M Rivera-Hoyos, Ángela M Cardozo-Bernal, Raúl A Poutou-Piñales, Aura M Pedroza-Rodríguez, Dennis J Díaz-Rincón, Alexander Rodríguez-López, Carlos J Alméciga-Díaz, Claudia L Cuervo-Patiño
Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations...
2017: Enzyme Research
https://www.readbyqxmd.com/read/28419821/expression-of-the-enzymatically-active-legumain-like-cysteine-proteinase-tvlegu-1-of-trichomonas-vaginalis-in-pichia-pastoris
#6
Gerardo Reséndiz-Cardiel, Rossana Arroyo, Jaime Ortega-López
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter...
April 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28416076/sweat-allergy-extrinsic-or-intrinsic
#7
REVIEW
Takaaki Hiragun, Makiko Hiragun, Kaori Ishii, Takanobu Kan, Michihiro Hide
Sweat is an exacerbation factor in atopic dermatitis (AD) in all age groups. A body core temperature elevation with sweating triggers cholinergic urticaria (CholU). We recently reported that AD symptoms are improved by tannic acid-containing spray, which suppresses the basophil histamine release induced by semi-purified sweat antigen in vitro, and by showering, which removes antigens in sweat from the skin surface. Sweat contains small amount of proteins including proteases, protease inhibitors, and anti-microbial peptides...
March 9, 2017: Journal of Dermatological Science
https://www.readbyqxmd.com/read/28412353/crystal-structure-of-rna-polymerase-ii-from-komagataella-pastoris
#8
Haruhiko Ehara, Takashi Umehara, Shun-Ichi Sekine, Shigeyuki Yokoyama
RNA polymerase II (Pol II) is a 12-subunit protein complex that conducts the transcription of mRNA and some small RNAs. In this work, the crystal structure of Pol II from the methylotropic yeast Komagataella pastoris (Pichia pastoris) was determined. While the structure is highly homologous to that of Pol II from the budding yeast Saccharomyces cerevisiae, the stalk and clamp modules of the K. pastoris Pol II displayed large inward rotations, closing the central cleft to a greater extent than in the known S...
April 12, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28411891/recombinant-expression-of-thermobifida-fusca-e7-lpmo-in-pichia-pastoris-and-escherichia-coli-and-their-functional-characterization
#9
Kelly B Rodrigues, Jéssica K A Macêdo, Tallyta Teixeira, Jéssica S Barros, Ana C B Araújo, Fernanda P Santos, Betânia F Quirino, Bruno S A F Brasil, Thaís F C Salum, Patrícia V Abdelnur, Léia C L Fávaro
The discovery of lytic polysaccharides monooxygenases copper dependent (LPMOs) revolutionized the classical concept that the cleavage of cellulose is a hydrolytic process in recent years. These enzymes carry out oxidative cleavage of cellulose (and other polysaccharides), acting synergistically with cellulases and other hydrolases. In fact, LPMOs have the potential for increasing the efficiency of the lignocellulosic biomass conversion in biofuels and high value chemicals. Among a small number of microbial LPMOs that have been characterized, some LPMOs were expressed and characterized biochemically from the bacteria Thermobifida fusca, using the host Escherichia coli...
April 9, 2017: Carbohydrate Research
https://www.readbyqxmd.com/read/28410888/temperature-control-strategy-to-enhance-the-activity-of-yeast-inoculated-into-compost-raw-material-for-accelerated-composting
#10
Kiyohiko Nakasaki, Hidehira Hirai
The effects of inoculating the mesophilic yeast Pichia kudriavzevii RB1, which is able to degrade organic acids, on organic matter degradation in composting were elucidated. When model food waste with high carbohydrate content (C/N=22.3) was used, fluctuation in the inoculated yeast cell density was observed, as well as fluctuation in the composting temperature until day 5 when the temperature rose to 60°C, which is lethal for the yeast. After the decrease in yeast, acetic acid accumulated to levels as high as 20mg/g-ds in the composting material and vigorous organic matter degradation was inhibited...
April 11, 2017: Waste Management
https://www.readbyqxmd.com/read/28409469/experimental-systems-to-study-yeast-pexophagy
#11
Shun-Ichi Yamashita, Masahide Oku, Yasuyoshi Sakai, Yukio Fujiki
Peroxisome abundance is tightly regulated according to the physiological contexts, through regulations of both proliferation and degradation of the organelles. Here, we describe detailed methods to analyze processes for autophagic degradation of peroxisomes, termed pexophagy, in yeast organisms. The assay systems include a method for biochemical detection of pexophagy completion, and one for microscopic visualization of specialized membrane structures acting in pexophagy. As a model yeast organism utilized in studies of pexophagy, the methylotrophic yeast Komagataella phaffii (Pichia pastoris) is referred to in this chapter and related information on the studies with baker's yeast (Saccharomyces cerevisiae) is also included...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28400000/influence-of-growth-conditions-on-adhesion-of-yeast-candida-spp-and-pichia-spp-to-stainless-steel-surfaces
#12
Ružica Tomičić, Peter Raspor
An understanding of adhesion behavior of Candida and Pichia yeast under different environmental conditions is key to the development of effective preventive measures against biofilm-associated infection. Hence in this study we investigated the impact of growth medium and temperature on Candida and Pichia adherence using stainless steel (AISI 304) discs with different degrees of surface roughness (Ra = 25.20-961.9 nm), material typical for the food processing industry as well as medical devices. The adhesion of the yeast strains to stainless steel surfaces grown in Malt Extract broth (MEB) or YPD broth at three temperatures (7 °C, 37 °C, 43 °C for Candida strains and 7 °C, 27 °C, 32 °C for Pichia strains) was assessed by crystal violet staining...
August 2017: Food Microbiology
https://www.readbyqxmd.com/read/28399696/molecular-strategies-to-increase-the-levels-of-heterologous-transcripts-in-komagataella-phaffii-for-protein-production
#13
Luiza Cesca Piva, Maritza Ocampo Bentacur, Viviane Castelo Branco Reis, Janice Lisboa De Marco, Lidia Maria Pepe de Moraes, Fernando Araripe Gonçalves Torres
Komagataella phaffii (formerly Pichia pastoris) is a well-known fungal system for heterologous protein production in the context of modern biotechnology. To obtain higher protein titers in this system many researchers have sought to optimize gene expression by increasing the levels of transcription of the heterologous gene. This has been typically achieved by manipulating promoter sequences or by generating clones bearing multiple copies of the desired gene. The aim of this work is to describe how these different molecular strategies have been applied in K...
February 22, 2017: Bioengineered
https://www.readbyqxmd.com/read/28399159/effects-of-the-rgd-loop-and-c-terminus-of-rhodostomin-on-regulating-integrin-%C3%AE-iib%C3%AE-3-recognition
#14
Yao-Tsung Chang, Jia-Hau Shiu, Chun-Hao Huang, Yi-Chun Chen, Chiu-Yueh Chen, Yung-Sheng Chang, Woei-Jer Chuang
Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 ≥ α5β1, and the 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbβ3 mutant...
2017: PloS One
https://www.readbyqxmd.com/read/28389915/implementation-of-data-dependent-isotopologue-fragmentation-in-13-c-based-metabolic-flux-analysis
#15
Teresa Mairinger, Stephan Hann
A novel analytical approach based on liquid chromatography coupled to quadrupole time of flight mass spectrometry, employing data-dependent triggering for analysis of isotopologue and tandem mass isotopomer fractions of metabolites of the primary carbon metabolism was developed. The implemented QTOFMS method employs automated MS/MS triggering of higher abundant, biologically relevant isotopologues for generating positional information of the respective metabolite. Using this advanced isotopologue selective fragmentation approach enables the generation of significant tandem mass isotopomer data within a short cycle time without compromising sensitivity...
April 7, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28389350/process-development-for-production-and-purification-of-the-schistosoma-mansoni-sm14-antigen
#16
Leonardo Damasceno, Gerd Ritter, Carl A Batt
The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L(-1). Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter...
April 5, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28386988/a-new-proteinaceous-pathogen-associated-molecular-pattern-pamp-identified-in-ascomycete-fungi-induces-cell-death-in-solanaceae
#17
Barbara Franco-Orozco, Adokiye Berepiki, Olaya Ruiz, Louise Gamble, Lucie L Griffe, Shumei Wang, Paul R J Birch, Kostya Kanyuka, Anna Avrova
Pathogen-associated molecular patterns (PAMPs) are detected by plant pattern recognition receptors (PRRs), which gives rise to PAMP-triggered immunity (PTI). We characterized a novel fungal PAMP, Cell Death Inducing 1 (RcCDI1), identified in the Rhynchosporium commune transcriptome sampled at an early stage of barley (Hordeum vulgare) infection. The ability of RcCDI1 and its homologues from different fungal species to induce cell death in Nicotiana benthamiana was tested following agroinfiltration or infiltration of recombinant proteins produced by Pichia pastoris...
April 7, 2017: New Phytologist
https://www.readbyqxmd.com/read/28385833/genome-sequences-of-cyberlindnera-fabianii-65-pichia-kudriavzevii-129-and-saccharomyces-cerevisiae-131-isolated-from-fermented-masau-fruits-in-zimbabwe
#18
Irma M H van Rijswijck, Martijn F L Derks, Tjakko Abee, Dick de Ridder, Eddy J Smid
Cyberlindnera fabianii 65, Pichia kudriavzevii 129, and Saccharomyces cerevisiae 131 have been isolated from the microbiota of fermented masau fruits. C. fabianii and P. kudriavzevii especially harbor promising features for biotechnology and food applications. Here, we present the draft annotated genome sequences of these isolates.
April 6, 2017: Genome Announcements
https://www.readbyqxmd.com/read/28382032/production-of-chimeric-acidic-%C3%AE-amylase-by-the-recombinant-pichia-pastoris-and-its-applications
#19
Deepak Parashar, Tulasi Satyanarayana
Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28376863/pichia-pastoris-versus-saccharomyces-cerevisiae-a-case-study-on-the-recombinant-production-of-human-granulocyte-macrophage-colony-stimulating-factor
#20
Anh-Minh Tran, Thanh-Thao Nguyen, Cong-Thuan Nguyen, Xuan-Mai Huynh-Thi, Cao-Tri Nguyen, Minh-Thuong Trinh, Linh-Thuoc Tran, Stephanie P Cartwright, Roslyn M Bill, Hieu Tran-Van
BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S...
April 4, 2017: BMC Research Notes
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