Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal a-Amylase Gene in Aspergillus oryzae

( Yanchen Yin ),( Youzhi Mao ),( Xiaolie Yin ),( Bei Gao ),( Dongzhi Wei ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.7

The filamentous fungus Aspergillus oryzae is a well-known expression host used to expresshomologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method.As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. ThePcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

Characterization of Cell Viability in Phaeocystis globosa Cultures Exposed to Marine Algicidal Bacteria

Xiaoli Hu,Pinghe Yin,Ling Zhao,Qiming Yu 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.1

The bloom of Phaeocystis globosa has occurredfrequently in the southern coastal areas of China in recentyears, which has led to substantial economic losses. Thisstudy investigated the effects of culture broth of strains Y1and Y4 isolated from algal blooms in Zhuhai, China onphysiological characteristics and cell viability of P. globosa. The increase in the levels of reactive oxygen species (ROS)in P. globosa cells exposed to strains Y1 and Y4 culturebroth were detected, indicating that the algal cells sufferedfrom oxidative damage. The surplus ROS induced theincrease of malondialdehyde (MDA) contents and theactivities of antioxidant enzymes, including superoxidedismutase (SOD) and catalase (CAT). The decrease inprotein content indicated that strains Y1 and Y4 culturebroth inhibited cell growth. The contents of pigmentsdecreased after 96 h treatment, indicated that oxidative stressdestroyed pigment synthesis. Furthermore, flow cytometrycoupled with the propidium iodide stain and chlorophyllauto-fluorescence was used to investigate cell viability. Results showed that chlorophyll fluorescence intensitiesand cell integrity decreased with time of exposure, whichdemonstrated that strains Y1 and Y4 culture broth couldchange membrane permeability and resulted in the loss ofthese photosynthetic pigments. The isolated strains wereidentified as Bacillus sp. by culture morphology, biochemicalreactions, and homology research based on 16S rDNA. Overall, these findings suggested that oxidative stress causedfrom Bacillus sp. potentially destroyed pigment synthesisand cell membrane integrity, and ultimately led to the lysisof the algal cells.

The Prebiotic Lactosucrose Modulates Gut Metabolites and Microbiota in Intestinal Inflammatory Rats

Xiaoli Zhou,Zheng Ruan,Xiaoliu Huang,Yan Zhou,Shiqiang Liu,Yulong Yin 한국식품과학회 2014 Food Science and Biotechnology Vol.23 No.1

The effect of lactosucrose on the short chainfatty acid (SCFA) content, the intestinal flora, the acidity,and the amino nitrogen (NH3-N) content of the intestine ininflammatory bowel disease (IBD) rats was investigated. Dietary lactosucose supplementation increased amounts ofbeneficial bacteria and diminished amounts of pathogenicbacteria. Lactosucrose decreased the NH3-N content in cecaland colonic digesta, compared to inflammatory rats. Lactosucrose increased the acidity in the intestinal lumen,and the acetic acid, propionic acid, and butyric acidcontents in cecal and colonic digesta, compared toinflammatory rats. The butyrate content in the lactosucrosegroup was higher than for the inflammatory, sulfasalazine,and normal groups. Lactosucrose promoted beneficialintestinal health and prevented intestinal inflammation.

Enhanced Microwave Absorption Properties of CeO2 Nanoparticles Supported on Reduced Graphene Oxide

Qing Yin,Honglong Xing,Ruiwen Shu,Xiaoli Ji,Dexin Tan,Ying Gan 성균관대학교(자연과학캠퍼스) 성균나노과학기술원 2016 NANO Vol.11 No.5

In this work, reduced graphene oxide/CeO2 nanocomposites (RGO/CeO2) with two different RGO contents were synthesized using a facile one-step hydrothermal method, and the microwave absorption properties of RGO/CeO2 were investigated for the first time. Morphology and structure analysis shows that the CeO2 nanoparticles are uniformly dispersed on the RGO sheets with average size of 15 nm. The as-prepared RGO/CeO2 exhibits excellent microwave absorbability. An optimal reflection loss (RL) of -32 dB is found at 17 GHz with a coating layer thickness of 1.5 mm. The product with a coating layer thickness of only 2.0 mm shows a bandwidth of 4.3 GHz, corresponding to RL at -10 dB (90% of electromagnetic wave absorption). Compared with pristine RGO or pure CeO2 nanoparticles, the reported nanocomposites achieved both wider and stronger wave absorption in the frequency range of 2–18 GHz. The enhanced microwave absorption properties are attributed to the conductive loss and dielectric loss mainly caused by the higher oxygen vacancy concentration of CeO2 in RGO/CeO2, which is demonstrated by X-ray photoelectron spectroscopy. Moreover, multiple interfacial polarizations occurring in the multi-interfaces between CeO2 and RGO sheets may be beneficial to microwave absorption. RGO/CeO2 could be used as an attractive candidate for the new type of microwave absorptive materials.

Photosynthetic inhibition and oxidative stress to the toxic Phaeocystis globosa caused by a diketopiperazine isolated from products of algicidal bacterium metabolism

Shuo Tan,Xiaoli Hu,Pinghe Yin,Ling Zhao 한국미생물학회 2016 The journal of microbiology Vol.54 No.5

Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine- 1,4-dione, cyclo-(Pro-Gly), by GC-MS and 1H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.

Kinetin inhibits apoptosis of aging spleen cells induced by D-galactose in rats

Mengyun Li,Wu-Qing OuYang,Xiaoli Wu,Yin Zheng,Yunpeng Wei,Lei An 대한수의학회 2014 JOURNAL OF VETERINARY SCIENCE Vol.15 No.3

Kinetin (Kn) is a cytokinin growth factor that exertsseveral anti-aging and antioxidant effects on cells andorgans. To investigate the mechanism underlying apoptoticevents in aging cells induced by D-galactose (D-gal), weexamined the effect of Kn delivered via nuchalsubcutaneous injection on D-gal-induced aging andapoptosis in rats. Our results showed that interleukin (IL)-2levels and mitochondrial membrane potential (Δ Ψ m) weredecreased by Kn in aging rats while IL-6 production andapoptosis increased. In addition, the expression ofanti-apoptotic Bcl-2 was low while that of Bax was high inthe aging group. After treated with Kn, compared withaging group, there showed obvious difference in Kn groupwith elevated IL-2, proliferation index, Bcl-2, Δ Ψ m anddecreased IL-6 and Bax in splenic lymphocyte. Based onthese results, we concluded that Kn can effectively protectthe rat spleen from aging, apoptosis, and atrophy.

Preclinical investigation of artesunate as a therapeutic agent for hepatocellular carcinoma via impairment of glucosylceramidase-mediated autophagic degradation

Chen Wenjia,Ma Zhaochen,Yu Lingxiang,MAO Xia,Ma Nan,Guo Xiaodong,Yin Xiaoli,Jiang Funeng,Wang Qian,Wang Jigang,Fang Mingliang,Lin Na,Zhang Yanqiong 생화학분자생물학회 2022 Experimental and molecular medicine Vol.54 No.-

Artesunate (ART) has been indicated as a candidate drug for hepatocellular carcinoma (HCC). Glucosylceramidase (GBA) is required for autophagic degradation. Whether ART regulates autophagic flux by targeting GBA in HCC remains to be defined. Herein, our data demonstrated that the dramatic overexpression of GBA was significantly associated with aggressive progression and short overall survival times in HCC. Subsequent experiments revealed an association between autophagic activity and GBA expression in clinical HCC samples, tumor tissues from a rat model of inflammation-induced HCC and an orthotopic mouse model, and human HCC cell lines. Interestingly, probe labeling identified GBA as an ART target, which was further verified by both a glutathione-S-transferase pulldown assay and surface plasmon resonance analysis. The elevated protein expression of LC3B, the increased numbers of GFP-LC3B puncta and double-membrane vacuoles, and the enhanced expression of SQSTM1/p62 indicated that the degradation of autophagosomes in HCC cells was inhibited by ART treatment. Both the in vitro and in vivo data revealed that autophagosome accumulation through targeting of GBA was responsible for the anti-HCC effects of ART. In summary, this preclinical study identified GBA as one of the direct targets of ART, which may have promising potential to inhibit lysosomal autophagy for HCC therapy.