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Farideh Siavoshi, Marzieh Sahraee, Hoda Ebrahimi, Abdolfatah Sarrafnejad, Parastoo Saniee
BACKGROUND: For controlling Helicobacter pylori infection in humans, its environmental reservoir should be determined. In this study, yeast isolates from an isolated village in Iran were studied for the intracellular occurrence of H. pylori. MATERIALS AND METHODS: In this study, yeasts were isolated from 29 samples, including oral swabs from villagers (n = 7), flowers and fruits (n = 6), honey and honeybees (n = 12) and miscellaneous samples (4). Yeasts were classified into 12 RFLP groups and identified by amplification of 26S rDNA and sequencing...
February 19, 2018: Helicobacter
Xindong Teng, Xiaoguang Chen, Ke Zhu, Hefei Xu
Objectives: Heparin-binding hemagglutinin (HBHA), a mycobacterial cell surface protein, mediates adhesion to nonphagocytic cells and the dissemination of Mycobacterium tuberculosis ( M. tuberculosis ) from the site of primary infection. Superior expression systems are required to obtain abundant M. tuberculosis proteins for the purpose of diagnosing M. tuberculosis infection or for the immunization. Here, HBHA was expressed by Pichia pastoris ( P. pastoris ) GS115 strain , and the immunogenicity of HBHA was evaluated...
February 2018: Iranian Journal of Basic Medical Sciences
Fernanda Gobbi Amorim, Johara Boldrini-França, Karla de Castro Figueiredo Bordon, Iara Aimê Cardoso, Edwin De Pauw, Loïc Quinton, Simone Kashima, Eliane Candiani Arantes
In general, hyaluronidases have a broad potential application on medicine and esthetics fields. Hyaluronidases from animal venoms cleave hyaluronan present in the extracellular matrix, acting as spreading factors of toxins into the tissues of the victim. However, the in-depth characterization of hyaluronidase from animal venoms has been neglected due to its instability and low concentration in the venom, which hamper its isolation. Thus, heterologous expression of hyaluronidase acts as a biotechnological tool in the obtainment of enough amounts of the enzyme for structural and functional studies...
February 17, 2018: Applied Microbiology and Biotechnology
Mariana Chesini, Evelyn Wagner, Diego J Baruque, Carolina E Vita, Sebastián F Cavalitto, Pablo D Ghiringhelli, Natalia L Rojas
Exoinulinases-enzymes extensively studied in recent decades because of their industrial applications-need to be produced in suitable quantities in order to meet production demands. We describe here the production of an acid-stable recombinant inulinase from Aspergillus kawachii in the Pichia pastoris system and the recombinant enzyme's biochemical characteristics and potential application to industrial processes. After an appropriate cloning strategy, this genetically engineered inulinase was successfully overproduced in fed-batch fermentations, reaching up to 840 U/ml after a 72-h cultivation...
February 15, 2018: Protein Expression and Purification
Liangcheng Jiao, Qinghua Zhou, Zhixin Xu, Li Xu, Yunjun Yan
Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy...
February 13, 2018: Protein Expression and Purification
Zhiliang Yang, Zisheng Zhang
Background: 2,3-butanediol (2,3-BD) is a bulk platform chemical with various potential applications such as aviation fuel. 2,3-BD has three optical isomers: (2R, 3R)-, (2S, 3S)- and meso-2,3-BD. Optically pure 2,3-BD is a crucial precursor for the chiral synthesis and it can also be used as anti-freeze agent due to its low freezing point. 2,3-BD has been produced in both native and non-native hosts. Several pathogenic bacteria were reported to produce 2,3-BD in mixture of its optical isomers including Klebsiella pneumoniae and Klebsiella oxytoca ...
2018: Biotechnology for Biofuels
Meng Zhang, Qilin Yu, Chenpeng Xiao, Kai Zhang, Dan Zhang, Biao Zhang, Mingchun Li
The ability to adapt to environmental changes is the necessary strategy of cell survival. Spt23 is responsible for regulation of Δ-9 desaturase expression in Pichia pastoris. Disruption of SPT23 leads to a remarkable decrease in cellular unsaturated fatty acids. In this study, we found that deletion of SPT23 resulted in growth defect under high temperature culture conditions and heat treatment induced the expression of SPT23. By measuring expression changes of heat shock proteins, protein levels and cellular localization of Hsf1, it revealed that the sensitivity of spt23Δ to high temperature was independent of heat shock response...
February 13, 2018: FEMS Yeast Research
Diego Gil de Los Santos, João Rodrigo Gil de Los Santos, Carlos Gil-Turnes, Giana Gaboardi, Luiza Fernandes Silva, Rodrigo França, Cristina Gevehr Fernandes, Fabricio Rochedo Conceição
In a previous paper we showed that the yeast Pichia pastoris X-33 grown in parboiled rice effluent supplemented with glycerol byproduct from the biodiesel industry improved the quality of the effluent. In this paper we show the validation of this yeast (PPE) as probiotic for broilers. Its effect on feed efficiency and immunomodulation was compared with the same yeast grown in yeast peptone dextrose medium (PPY), with Saccharomyces boulardii (SBY) and with the controls fed unsupplemented feed (CON). One-day-old female chicks were vaccinated against infectious bursal disease (IBD) and the titers of anti-IBD antibodies were measured by ELISA...
2018: PloS One
Xiutao Chen, Weiguang Li, Peng Ji, Yang Zhao, Chengyao Hua, Chao Han
Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme's commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis...
February 13, 2018: Scientific Reports
Dianhui Wu, Guolin Cai, Xiaomin Li, Bing Li, Jian Lu
OBJECTIVES: To optimize the expression of type A ferulic acid esterase (FaeA) from Aspergillus niger in Pichia pastoris X-33 using codon optimization. RESULTS: Recombinant FaeA was purified from the fermentation broth, with the maximum specific activity of 48.4 ± 0.1 U mg-1. Adding it during mashing process for beer brewing raised the filtration rate by 14.5% while the turbidity and viscosity declined by 22 and 6.9%, respectively. Addition of FaeA increased the concentrations of free ferulic acid (FA) and arabinoxylan (AX) in the wort, while the polymeric arabinoxylans content declined significantly...
February 12, 2018: Biotechnology Letters
Alessandra Luchini, Robin Delhom, Bruno Demé, Valérie Laux, Martine Moulin, Michael Haertlein, Harald Pichler, Gernot A Strohmeier, Hanna Wacklin, Giovanna Fragneto
The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way...
February 5, 2018: Colloids and Surfaces. B, Biointerfaces
Yan-Xiao Li, Ping Yi, Jun Liu, Qiao-Juan Yan, Zheng-Qiang Jiang
An engineered β-mannanase (mRmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris. Through high cell density fermentation, the expression level of mRmMan5A reached 79,680 U mL-1. The mRmMan5A showed maximum activity at pH 4.5 and 65 °C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200 °C for 7.5 min, and then hydrolyzed by mRmMan5A. As a result, the total manno-oligosaccharide yield reached 34...
February 1, 2018: Bioresource Technology
Qiangqiang Huai, Luqiang Jia, Jian Ding, Shanshan Chen, Jiaowen Sun, Zhongping Shi
In heterologous protein productions by Pichia pastoris, methanol induction is generally initiated when cell density reaches very high level. However, this traditional strategy suffers with the problems of difficulty in DO control, toxic by-metabolites accumulation and low targeted protein titer. Therefore, initiating methanol induction at lower cell concentration is considered as an alternative strategy to overcome those problems. However, the methanol/energy regulation mechanisms of initiating induction at lower concentration are not clear and seldom reported...
February 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Yuying Sun, Jiquan Zhang, Jianhai Xiang
Chitin deacetylase (CDA, EC, belonging to a family of extracellular chitin-modifying enzymes, can catalyze the deacetylation of chitin. In this study, the full-length cDNA sequence encoding chitin deacetylase 1 (EcCDA1) was obtained from the Exopalaemon carinicauda. The complete nucleotide sequence of EcCDA1 contained a 1611 bp open reading frame (ORF) encoding EcCDA1 precursor of 536 amino acids. The domain architecture of the deduced EcCDA1 protein contained a signal peptide, a chitin-binding peritrophin-A domain (ChtBD2), a low-density lipoprotein receptor class A domain (LDLa) and a Polysacc_deac_1 domain...
February 5, 2018: Fish & Shellfish Immunology
Zahra Bahadori, Hamid Reza Kalhor, Seyed Javad Mowla
Keratinocyte Growth Factor (KGF) is a paracrine-acting, epithelial mitogen that plays a prominent role in the regeneration of damaged epithelial tissues. In spite of different attempts to produce recombinant human KGF in many organisms, including bacteria, mammalian cells, plant cells and insect cells; production of recombinant form suffers from lower yields and recovery relative to other recombinant proteins of similar size and properties. Due to many advantages of Pichia pastoris expression systems for producing industrial enzymes and pharmaceutical proteins, in this study P...
April 2018: Enzyme and Microbial Technology
Matthias Engleder, Melissa Horvat, Anita Emmerstorfer-Augustin, Tamara Wriessnegger, Stefanie Gabriel, Gernot Strohmeier, Hansjörg Weber, Monika Müller, Iwona Kaluzna, Daniel Mink, Martin Schürmann, Harald Pichler
Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F...
2018: PloS One
Vida Eraghi, Abdollah Derakhshandeh, Arsalan Hosseini, Azar Motamedi-Boroojeni
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants and there has been a shift in the public health approach to MAP and human diseases like Crohn's disease. The prevention of infection by MAP in ruminants is thought to deter the high impact of economic losses in the level of dairy industry and possible spreading of this pathogen in dairy products. The present study was done to investigate the construction and expression of the soluble form of a novel fusion protein, consisting of Heparin-binding hemagglutinin (HBHA) and high antigenic region of Fibronectin Attachment Protein-P (FAP-P), in order to introduce as a Th1 inducer subunit vaccine against MAP...
December 2017: Molecular Biology Research Communications
William P Sheffield, Louise J Eltringham-Smith, Varsha Bhakta
BACKGROUND/AIMS: The Kunitz Protease Inhibitor (KPI) domain of protease nexin 2 (PN2) potently inhibits coagulation factor XIa. Recombinant KPI has been shown to inhibit thrombosis in mouse models, but its clearance from the murine circulation remains uncharacterized. The present study explored the pharmacokinetic and pharmacodynamic effects of fusing KPI to human serum albumin (HSA) in fusion protein KPIHSA. METHODS: Hexahistidine-tagged KPI (63 amino acids) and KPIHSA (656 amino acids) were expressed in Pichia pastoris yeast and purified by nickel-chelate chromatography...
February 1, 2018: Cellular Physiology and Biochemistry
E Robledo-Leal, L G Rivera-Morales, M P Sangorrín, G M González, G Ramos-Alfano, J M Adame-Rodriguez, J M Alcocer-Gonzalez, E T Arechiga-Carvajal, C Rodriguez-Padilla
Although invasive infections and mortality caused by Candida species are increasing among compromised patients, resistance to common antifungal agents is also an increasing problem. We analyzed 60 yeasts isolated from patients with invasive candidiasis using a PCR/RFLP strategy based on the internal transcribed spacer (ITS2) region to identify different Candida pathogenic species. PCR analysis was performed from genomic DNA with a primer pair of the ITS2-5.8S rDNA region. PCR-positive samples were characterized by RFLP...
February 1, 2018: Brazilian Journal of Biology, Revista Brasleira de Biologia
Yuanyuan Zhu, Lu Wu, Junjun Zhu, Yong Xu, Qiang Yong, Shiyuan Yu
To obtain a global view of the dynamic phospholipids in Pichia stipitis during the ethanol fermentation in the presence of three representative inhibitors (vanillin, 5-hydroxymethylfurfural, and acetic acid). Considerable efforts have been expended to elucidate the biochemical mechanisms of inhibitors interaction with phospholipids. In this study, a comparative lipidomic analysis was performed using liquid chromatography-mass/mass spectrometry (LC-MS/MS) on P. stipitis. Partial least squares-discriminate analysis (PLS-DA) was used to deal with the large quantity of data generated using the systematic methods...
February 1, 2018: Biochemical and Biophysical Research Communications
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