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Yufeng Qin, Yaosheng Chen, Zhiguo Liu, Ying Zhang, Hailong Liu, Zuyong He
A mammalian nonclassical secretion sequence derived from mouse Engrailed2 homeoprotein (En2) was used to direct the secretion of the enhanced green fluorescent protein from Pichia pastoris. This signal peptide conferred the transport of enhanced green fluorescent protein into periplasm through an endoplasmic reticulum-golgi independent pathway, without inducing severe unfolded protein response as compared with Saccharomyces cerevisae α-factor preprosequence. This study implies that this mammalian nonclassical signal peptide could be developed as a useful tool for delivering cargoes to the cell surface of yeast...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Renhui Bai, Yunbo Zhang, Chundi Wang, Feiyang Zhang, Zhe Zhang, Fubao Sun, Zhenyu Zhang
Deficient activity of endo-1,4-beta-glucanase II (Cel5A) secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. Therefore, we expressed Cel5A in Pichia pastoris by constructing a recombinant strain. With the gene optimization based on codon bias, and the construction of expression vector pPIC9K-eg2, the optimized gene was electro-transformed into P. pastoris GS115 to form transformants. Then, a high Cel5A activity producing recombinant, namely P...
October 25, 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Zhangcai Luo, Jing Miao, Wei Luo, Guoying Li, Yao Du, Xiaobin Yu
OBJECTIVE: To explore an efficient use of crude glycerol for the production of a highly thermostable β-mannanase (ReTMan26) by Pichia pastoris X33. RESULTS: Cell growth was significantly inhibited by 4 and 6% (w/v) crude glycerol in 250 ml shake-flasks and in 5 l bioreactor batch cultures, respectively, but not affected by pure glycerol at the same concentrations. For further study, the impact of various impurities in crude glycerol on the cell growth of, and ReTMan26 production by, Pichia pastoris was investigated...
October 12, 2017: Biotechnology Letters
Kristína Markošová, Andrea Camattari, Michal Rosenberg, Anton Glieder, Nicholas J Turner, Martin Rebroš
OBJECTIVE: To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production. RESULTS: Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 Mut(S) strain for the first time. Various transformants were screened at microscale level...
October 10, 2017: Biotechnology Letters
Xue Zhang, Shuiying Yang, Xinqiang Li, Pei Zhu, Enyu Xie, Zhenlun Li
The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could specifically dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. The CQMa102 phosphatase was expressed in Pichia pastoris to verify its protease activity. The molecular weight (MW) and the isoelectric point (pI) of the phosphatase were about 85 kDa and 6...
October 11, 2017: Bioscience, Biotechnology, and Biochemistry
Hao Tan, Jie Tang, Xiaolin Li, Tianhai Liu, Renyun Miao, Zhongqian Huang, Yong Wang, Bingcheng Gan, Weihong Peng
Psychrophilic phytases suitable for aquaculture are rare. In this study, a phytase of histidine acidic phosphatase (HAP) family was identified in Morchella importuna, a psychrophilic mushroom. The phytase showed 38% identity with Aspergillus niger PhyB, which was the closest hit. The M. importuna phytase was overexpressed in Pichia pastoris, purified and characterized. The phytase had an optimum temperature at 25°C, which is the lowest among all the known phytases to our knowledge. The optimum pH (6.5) is higher than most of the known HAP phytases, which is fit for the weak acidic condition in fish gut...
October 11, 2017: Journal of Microbiology and Biotechnology
Raimondo Gaglio, Antonio Alfonzo, Nicola Francesca, Onofrio Corona, Rosalia Di Gerlando, Pietro Columba, Giancarlo Moschetti
The "Spiritu re fascitrari" (SRF) is a typical Sicilian distillate obtained from the by-products of traditional process of honey production. Although some alcoholic fermentation of honey based products have been described, the present research represents the first investigation on the yeast ecology and the physico-chemical characteristics of honey by-products subjected to an alcoholic fermentation followed by distillation. All samples collected during manufacturing process were analysed for the count of total, osmophilic and osmotolerant yeasts...
September 11, 2017: International Journal of Food Microbiology
Chiemi Imaizumi, Harumi Tomatsu, Kiminari Kitazawa, Yoshihisa Yoshimi, Seiji Shibano, Kaoru Kikuchi, Masatoshi Yamaguchi, Satoshi Kaneko, Yoichi Tsumuraya, Toshihisa Kotake, Björn Usadel
The major plant sugar l-arabinose (l-Ara) has two different ring forms, l-arabinofuranose (l-Araf) and l-arabinopyranose (l-Arap). Although l-Ara mainly appears in the form of α-l-Araf residues in cell wall components, such as pectic α-1,3:1,5-arabinan, arabinoxylan, and arabinogalactan-proteins (AGPs), lesser amounts of it can also be found as β-l-Arap residues of AGPs. Even though AGPs are known to be rapidly metabolized, the enzymes acting on the β-l-Arap residues remain to be identified. In the present study, four enzymes, which we call β-l-ARAPASE (APSE) and α-GALACTOSIDASE 1 (AGAL1), AGAL2, and AGAL3, are identified as those enzymes that are likely to be responsible for the hydrolysis of the β-l-Arap residues in Arabidopsis thaliana...
July 20, 2017: Journal of Experimental Botany
Luqiang Jia, Tingyong Tu, Qiangqiang Huai, Jiaowen Sun, Shanshan Chen, Xin Li, Zhongping Shi, Jian Ding
In heterologous protein productions by P. pastoris, methanol induction is generally initiated when cell concentration reaches very high density. The alternative strategy by initiating methanol induction at lower cells concentration was also reported to be effective in easing DO control, reducing toxic by-metabolites accumulation and increasing targeted proteins titers. However, the methanol/energy regulation mechanisms are seldom reported. We theoretically analyzed the methanol/energy metabolisms in protein expression process with the strategies of initiating induction at higher or lower cells concentrations, using monellin production as a prototype...
2017: PloS One
Nannan Zhao, Yanshan Xu, Kuang Wang, Suiping Zheng
We explored the ability of an Aspergillus niger α-glucosidase displayed on P. pastoris to act as a whole-cell biocatalyst (Pp-ANGL-GCW61) system to synthesize isomalto-oligosaccharides (IMOs). IMOs are a mixture that includes isomaltose (IG2), panose (P) and isomaltotriose (IG3). In this study, the IMOs were synthesized by a hydrolysis-transglycosylation reaction in an aqueous system of maltose. In a 2-mL reaction system, the IMOs were synthesized with a conversion rate of approximately 49% in 2 h when 30% maltose was utilized under optimal conditions by Pp-ANGL-GCW61...
October 5, 2017: Journal of Agricultural and Food Chemistry
Svetlana A Korban, Kirill S Bobrov, Maria A Maynskova, Stanislav N Naryzhny, Olga L Vlasova, Elena V Eneyskaya, Anna A Kulminskaya
No abstract text is available yet for this article.
August 1, 2017: Protein Engineering, Design & Selection: PEDS
Maria Teresa P Gonçalves Dos Santos, María José Benito, María de Guía Córdoba, Nuno Alvarenga, Santiago Ruiz-Moyano Seco de Herrera
This study investigated the yeast community present in the traditional Portuguese cheese, Serpa, by culture-dependent and -independent methods. Sixteen batches of Serpa cheeses from various regional industries registered with the Protected Designation of Origin (PDO) versus non-PDO registered, during spring and winter, were used. Irrespective of the producer, the yeast counts were around 5log CFU/g in winter and, overall, were lower in spring. The yeast species identified at the end of ripening (30days), using PCR-RFLP analysis and sequencing of the 26S rRNA, mainly corresponded to Debaryomyces hansenii and Kluyveromyces marxianus, with Candida spp...
December 4, 2017: International Journal of Food Microbiology
Haoyu Zhou, Chengming Wang
Fatty acid desaturation enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids. ω-3 desaturase has an important role in converting ω-6 fatty acids into ω-3 fatty acids. Although genes for this desaturase have been identified, the enzymatic activity of Δ-17 with or without transmembrane domain, and the function of the Δ-17 desaturase is poorly understood. In the present study, a transgenic microorganism was used to clone the Δ-17 full length (Δ-17FL) and Δ-17 without transmembrane domain (Δ-17NT), the expression efficiency was improved and western blotting was used to detect the protein expression level...
September 2017: Experimental and Therapeutic Medicine
Ding Li, Bo Zhang, Shuting Li, Jie Zhou, Hui Cao, Yan Huang, Zhongli Cui
Pichia pastoris is widely used as a platform for heterologous protein expression because of its high volumetric productivity. Multicopy integration of the target gene is commonly used to improve the production of the target protein. Cre/lox recombination system is a powerful tool for the marker rescue during multiple integrations with one selection marker. Here we reported a novel expression vector based on the Cre/lox recombination system for multiple integrations of target gene to construct multicopy expression strain of P...
2017: Frontiers in Microbiology
Lin Wu, Huijun Li, Tianle Tang
Fluorescence resonance energy transfer substrates of sortase A are too expensive to be used to roughly screen high-throughput sortase A inhibitors. This makes therapeutic strategies difficult to realize in a clinical therapeutic use. Instead, we design here an LPETG-EGFP (leucine, proline, glutamic, threonine and glycine-enhanced green fluorescence) protein displayed on a yeast surface as a substrate by adaptively reducing the cost. We do this by optimizing the induction conditions of sortase A expression in Escherichia coli DE3(BL21) and catalyzing LPETG proteins, which are displayed on surface of Pichia pastoris...
January 24, 2017: Bioengineering
Neil Pj Price, Trina M Hartman, Jiakun Li, Kiran K Velpula, Todd A Naumann, Maheedhara R Guda, Biao Yu, Kenneth M Bischoff
Tunicamycins (TUN) are inhibitors of the UDP-HexNAc: polyprenol-P HexNAc-1-P transferase family of enzymes, which initiate the biosynthesis of bacterial peptidoglycan and catalyze the first step in eukaryotic protein N-glycosylation. The TUN are therefore general and potent toxins to both eukaryotes and prokaryotes. Screening a library of synthetic TUN against Bacillus and yeast identified TUN that are antibacterial, but have significantly reduced eukaryotic toxicity. One of these (Tun-15:0) differs from the native TUN control only by the lack of the conjugated double bond in the tunicaminyl N-acyl group...
September 27, 2017: Journal of Antibiotics
Yi Yang, Ning Zhu, Jinshui Yang, Yujian Lin, Jiawen Liu, Ruonan Wang, Fengqin Wang, Hongli Yuan
BACKGROUND: Xylan, the major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-L-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes...
September 26, 2017: Microbial Cell Factories
Huan Chen, Cui Cao, Anna Kulinich, Li Liu, Yong-Sam Jung, Josef Voglmeir
AIM AND OBJECTIVE: This study describes the design and evaluation of an expression vector for Pichia pastoris (pPICZαBHF), which is based on the commercial vector construct pPICZαB. MATERIAL AND METHOD: The performance of pPICZαBHF was evaluated with red fluorescence protein as a reporter. Additional His- and Flag-tags on the N-terminal ensure a simplified protein purification procedure. Transformation efficiency, expression level and plasmid maintenance were studied in order to test the functionality and usefulness of the constructed vector...
September 25, 2017: Combinatorial Chemistry & High Throughput Screening
Yun-Peng Bai, Xiao-Jing Luo, Yu-Lian Zhao, Chun-Xiu Li, Dian-Sheng Xu, Jian-He Xu
The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPHM9, an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 gprotein/L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L(-1) h(-1). PoOPHM9 displayed its high activity and stability in the presence of 0...
October 9, 2017: Journal of Agricultural and Food Chemistry
Yunfeng Zhang, Hao Huang, Xinhui Yao, Guocheng Du, Jian Chen, Zhen Kang
Streptomyces griseus trypsin (SGT) possesses enzymatic properties similar to mammalian trypsins and has great potential applications in the leather processing, bioethanol, detergent and pharmaceutical industry. Here, a new strategy was reported for improving its stable, active secretory production through engineering of its propeptide and self-degradation sites. By rationally introducing hydrophobic mutations into the N-terminus of SGT Exmt (R145I), replacing the propeptide with FPVDDDDK and engineering the α-factor signal peptide, trypsin production (amidase activity) was improved to 177...
August 4, 2017: Bioresource Technology
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