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Hitomi Nakamura, Naoko Oda-Ueda, Tadashi Ueda, Takatoshi Ohkuri
The production of therapeutic monoclonal antibodies is costly; therefore, antibody-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then the mutant H:L178 N Fab was expressed in Pichia pastoris...
June 14, 2018: Biochemical and Biophysical Research Communications
Mohamed Boumaiza, Haifa Chahed, Aymen Ezzine, Maryse Jaouen, Alessandra Gianoncelli, Giovanna Longhi, Fernando Carmona, Paolo Arosio, Marie-Agnès Sari, Mohamed Nejib Marzouki
No abstract text is available yet for this article.
July 2018: Journal of Molecular Recognition: JMR
Hyun Joo Park, Jung-Hoon Bae, Hyeok-Jin Ko, Sun-Hee Lee, Bong Hyun Sung, Jong-In Han, Jung-Hoon Sohn
Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low-pH production of D-lactic acid, an acid-tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain was able to grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing pyruvate decarboxylase 1 gene (PDC1) with D-lactate dehydrogenase gene (D-LDH) derived from Lactobacillus plantarum...
June 13, 2018: Biotechnology and Bioengineering
Yang Pan, Dan Wu, Jing Wu
Pichia pastoris is a versatile protein expression system. At present, the expression plasmids are integrated plasmids whereas the episomal plasmids are rarely used. In this study, the autonomously replicating sequence derived from yeast itself was ligated into the integrative expression vector pGAP to generate an autonomously replicative expression vector pGAPZαA-PARS. When the vector was used to express the xylanase (XynA) gene in P. pastoris, the highest enzyme activity reached 343 U/mL with glycerol as the carbon source, which was 45...
May 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Lei Shi, Xiaolong Wang, Jinjia Wang, Ping Zhang, Fei Qi, Menghao Cai, Yuanxing Zhang, Xiangshan Zhou
Two catabolite repressor genes (MIG1 and MIG2) were previously identified in Pichia pastoris, and the derepression of alcohol oxidase (AOX) expression was realized in Δmig1 or Δmig1Δmig2 mutants grown in glycerol, but not in glucose. In this study, genome-wide RNA-seq analysis of Δmig1Δmig2 and the wild-type strain grown in glycerol revealed that the expression of numerous genes was greatly altered. Nearly 7% (357 genes) of approximately 5276 genes annotated in P. pastoris were significantly upregulated, with at least a two-fold differential expression in Δmig1Δmig2; the genes were mainly related to cell metabolism...
April 2018: Genes & Genomics
Erika T da Cruz Almeida, Isabella de Medeiros Barbosa, Josean F Tavares, José M Barbosa-Filho, Marciane Magnani, Evandro L de Souza
This study evaluated the efficacy of the essential oil from Mentha spicata L. (MSEO) and M . × villosa Huds. (MVEO) to inactivate Candida albicans, C. tropicalis, Pichia anomala and Saccharomyces cerevisiae in Sabouraud dextrose broth and cashew, guava, mango, and pineapple juices during 72 h of refrigerated storage. The effects of the incorporation of an anti-yeast effective dose of MSEO on some physicochemical and sensory characteristics of juices were evaluated. The incorporation of 3.75 μL/mL MSEO or 15 μL/mL MVEO caused a ≥5-log reductions in counts of C...
2018: Frontiers in Microbiology
Zeinab Bagheri, Hamide Ehtesabi, Zahra Hallaji, Hamid Latifi, Ebrahim Behroodi
Carbon dots (CDs) as a new fluorescent material with excellent water solubility, chemical inertness, and easy surface modification are a good candidate for bioimaging and biosensing due to their low toxicity and good biocompatibility. Although carbon is not an intrinsically toxic substance, carbon nanomaterials such as CDs may cause risks to human health and the potentially hazardous effects of CDs on various living systems must be completely determined. So far, cytotoxicity studies of CDs have focused on human cells and are mainly conducted on limited cell lines...
June 7, 2018: Ecotoxicology and Environmental Safety
Carmen Oeo-Santos, Salvador Mas, Sara Benedé, María López-Lucendo, Joaquín Quiralte, Miguel Blanca, Cristobalina Mayorga, Mayte Villalba, Rodrigo Barderas
The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software...
June 5, 2018: Journal of Proteomics
Francisco Javier De la Torre González, Daniel Oswaldo Gutiérrez Avendaño, Anne Christine Gschaedler Mathis, Manuel Reinhart Kirchmayr
Non- Saccharomyces yeasts are widespread microorganisms and some time ago were considered contaminants in the beverage industry. However, nowadays they have gained importance for their ability to produce aromatic compounds, which in alcoholic beverages improves aromatic complexity and therefore the overall quality. Thus, identification and differentiation of the species involved in fermentation processes is vital and can be classified in traditional methods and techniques based on molecular biology. Traditional methods, however, can be expensive, laborious and/or unable to accurately discriminate on strain level...
June 6, 2018: Rapid Communications in Mass Spectrometry: RCM
Yuzhou Wang, Rui Ma, Shigui Li, Mingbo Gong, Bin Yao, Yingguo Bai, Jingang Gu
Alkaline lipases with adaptability to low temperatures and strong surfactant tolerance are favorable for application in the detergent industry. In the present study, a lipase-encoding gene, TllipA, was cloned from Trichoderma lentiforme ACCC30425 and expressed in Pichia pastoris GS115. The purified recombinant TlLipA was found to have optimal activities at 50 °C and pH 9.5 and retain stable over the pH range of 6.0-10.0 and 40 °C and below. When using esters of different lengths as substrates, TlLipA showed preference for the medium length p-nitrophenyl octanoate...
June 5, 2018: AMB Express
Hana Raschmanová, Leona Paulová, Barbora Branská, Zdeněk Knejzlík, Karel Melzoch, Karin Kovar
Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris...
June 5, 2018: Folia Microbiologica
Ravi Kant Rajpoot, Rahul Shukla, Upasana Arora, Sathyamangalam Swaminathan, Navin Khanna
Dengue is a significant public health problem worldwide, caused by four antigenically distinct mosquito-borne dengue virus (DENV) serotypes. Antibodies to any given DENV serotype which can afford protection against that serotype tend to enhance infection by other DENV serotypes, by a phenomenon termed antibody-dependent enhancement (ADE). Antibodies to the viral pre-membrane (prM) protein have been implicated in ADE. We show that co-expression of the envelope protein of all four DENV serotypes, in the yeast Pichia pastoris, leads to their co-assembly, in the absence of prM, into tetravalent mosaic VLPs (T-mVLPs), which retain the serotype-specific antigenic integrity and immunogenicity of all four types of their monomeric precursors...
June 5, 2018: Scientific Reports
Rika Suzuki, Masayoshi Sakakura, Masaki Mori, Moe Fujii, Satoko Akashi, Hideo Takahashi
Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E...
June 5, 2018: Journal of Biomolecular NMR
Peter Morrison-Whittle, Soon A Lee, Bruno Fedrizzi, Matthew R Goddard
The products of microbial metabolism form an integral part of human industry and have been shaped by evolutionary processes, accidentally and deliberately, for thousands of years. In the production of wine, a great many flavor and aroma compounds are produced by yeast species and are the targets of research for commercial breeding programs. Here we demonstrate how co-evolution with multiple species can generate novel interactions through serial co-culture in grape juice. We find that after ~65 generations, co-evolved strains and strains evolved independently show significantly different growth aspects and exhibit significantly different metabolite profiles...
2018: Frontiers in Microbiology
Mei Liu, Ying Liang, Hui Zhang, Gangcheng Wu, Li Wang, Haifeng Qian, Xiguang Qi
During freezing process, ice crystals formation leads to the deterioration in physicochemical properties and networks of gluten proteins. The cryoprotective effects of recombinant carrot ( Daucus carota) antifreeze protein (rCaAFP), type II antifreeze protein from Epinephelus coioides (rFiAFP), and Tenebrio molitor antifreeze protein (rTmAFP) produced from Pichia pastoris GS115 on hydrated gluten, glutenin, and gliadin during freezing were investigated. The thermal hysteresis (TH) activity and ice crystals morphology modification ability of recombinant antifreeze proteins (rAFPs) were analyzed by differential scanning calorimetry (DSC) and cryomicroscope, respectively...
June 4, 2018: Journal of Agricultural and Food Chemistry
Takuya Koseki, Kyotaro Ichikawa, Katsuto Sasaki, Yoshihito Shiono
We report the characterization of tannase-encoding gene, AotanB, from Aspergillus oryzae and its recombinant enzyme expressed in Pichia pastoris. The gene except for the signal sequence was cloned into a vector pPICZαA and the recombinant protein was secreted into the medium as an active enzyme. Recombinant AoTanB highly expressed in the incubation at 18°C compared to 30°C. Purified recombinant protein exhibited smeared band with molecular mass of approximately 90-120 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis...
May 31, 2018: Journal of Bioscience and Bioengineering
Pengfei Zhang, Xianghua Yuan, Yuguang Du, Jian-Jun Li
BACKGROUND: Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P...
June 1, 2018: BMC Biotechnology
Shitarashmi Sahu, Krishna Pramanik
This paper investigates the efficiency of the organic acids on the pretreatment of an industrially generated cotton gin waste for the removal of lignin, thereby releasing cellulose and hemicellulose as fermentable sugar components. Cotton gin waste was pretreated with various organic acids namely lactic acid, oxalic acid, citric acid, and maleic acid. Among these, maleic acid was found to be the most efficient producing maximum xylose sugar (126.05 ± 0.74 g/g) at the optimum pretreatment condition of 150 °C, 500 mM, and 45 min...
June 2, 2018: Applied Biochemistry and Biotechnology
Andrea B Zepeda, Adalberto Pessoa, Jorge G Farías
Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable...
May 19, 2018: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
Chengyao Hua, Weiguang Li, Wei Han, Qunqing Wang, Pengsheng Bi, Chao Han, Lusheng Zhu
A new endoglucanase encoding gene (ctendo7) was cloned from the thermophilic fungus Chaetomium thermophilum and heterologously expressed in Pichia pastoris. The recombinant CTendo7 enzyme was purified by Ni2+ affinity chromatography and subsequently characterized. CTendo7 belongs to glycoside hydrolase family 7, and exhibited considerable activity against sodium carboxymethyl cellulose (CMC-Na) and xylan of 1.91 IU/mg and 3.05 IU/mg at the optimum reaction condition of 55 °C, pH 5.0, respectively. The purified enzyme displayed relatively good thermostability...
May 26, 2018: International Journal of Biological Macromolecules
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