Analysis of Phosphatidylinositol 3,4,5-Trisphosphates of PTEN Expression on Mammalian Cells

( Nusrat Jahan ),( Taeseong Park ),( Young Hwan Kim ),( Dongsun Lee ),( Hackyoung Kim ),( Kwangmo Noh ),( Young Jun Kim ) 한국질량분석학회 2013 Mass spectrometry letters Vol.4 No.3

The goal of this study is to find an experimental condition which enables us to perform enzymatic studies on the cellular behavior of PTEN (phosphatase and tensine homolog) through identification of molecular species of phosphatidylinositol 3,4,5- trisphosphates and their quantitative analysis in a mammalian cell line using mass spectrometry. We initially exployed a two-step extraction process using HCl for extraction of phosphatidylinositol 3,4,5-trisphosphates from two mammalian cell lines and further analyzed the extracted phosphatidylinositol 3,4,5-trisphosphates using tandem mass spectrometry for the identification of them. We finally quantified the concentration of phosphatidylinositol 3,4,5-trisphosphates using internal standard calibration. From these observation, we found that HEK 293-T cells is a good model to examine the enzymatic behavior of PTEN in a cell, and the minimum amount of phosphatidylinositol 3,4,5-trisphosphates is more than 50 pmol for quantification in a mass spectrometer. These results suggest that the well-optimized experimental conditions are required for the investigation of the cellular PTEN in terms of the catalytic mechanism and further for the detailed identification of cellular substrates.

Analysis of Phosphatidylinositol 3,4,5-Trisphosphates of PTEN Expression on Mammalian Cells

Jahan, Nusrat,Park, Taeseong,Kim, Young Hwan,Lee, Dongsun,Kim, Hackyoung,Noh, Kwangmo,Kim, Young Jun Korean Society for Mass Spectrometry 2013 Mass spectrometry letters Vol.4 No.3

The goal of this study is to find an experimental condition which enables us to perform enzymatic studies on the cellular behavior of PTEN (phosphatase and tensine homolog) through identification of molecular species of phosphatidylinositol 3,4,5-trisphosphates and their quantitative analysis in a mammalian cell line using mass spectrometry. We initially exployed a two-step extraction process using HCl for extraction of phosphatidylinositol 3,4,5-trisphosphates from two mammalian cell lines and further analyzed the extracted phosphatidylinositol 3,4,5-trisphosphates using tandem mass spectrometry for the identification of them. We finally quantified the concentration of phosphatidylinositol 3,4,5-trisphosphates using internal standard calibration. From these observation, we found that HEK 293-T cells is a good model to examine the enzymatic behavior of PTEN in a cell, and the minimum amount of phosphatidylinositol 3,4,5-trisphosphates is more than 50 pmol for quantification in a mass spectrometer. These results suggest that the well-optimized experimental conditions are required for the investigation of the cellular PTEN in terms of the catalytic mechanism and further for the detailed identification of cellular substrates.

Catalysis Reaction Engineering, Industrial, Chemistry : Determination of Multiple Steady States in Oxidation of Monophenols by Tyrosinase with Enzymatic-Enzymatic-Chemical Model

( Guo Syong Chuang ),( An Chong Chao ),( Hsing Ya Li ) 한국화학공학회 2004 Korean Journal of Chemical Engineering Vol.21 No.5

Thermo-reversible injectable gel based on enzymatically-chopped low molecular weight methylcellulose for exenatide and FGF 21 delivery to treat types 1 and 2 diabetes

Kim, J.K.,Yoo, C.,Cha, Y.H.,Kim, Y.H. Elsevier Science Publishers 2014 Journal of controlled release Vol.194 No.-

Diabetes is the fastest growing metabolic disease that fails to utilize glucose properly due to insulin deficiency or insulin resistance. Although several limited studies demonstrated non-invasive means of protein delivery, major hurdles for commercial success such as short half-life, enzymatic degradation and low bioavailability still remain to overcome. Methylcellulose (MC), a hydrophobically-modified cellulose derivative, forms temperature reversible gel in aqueous solution. However, as the gelling temperature of MC is higher than body temperature, it should be lowered to below body temperature for practical clinical application. In order to decrease gelling temperature and increase bio-compatibility and bio-elimination of MC, the molecular weight of MC was decreased using enzymatic degradation method and confirmed by gel permeation chromatography. Bio-elimination of low molecular weight (LMw) MC was confirmed with non-invasive live image and ex vivo experiment. The exenatide and FGF 21 were physically loaded 100% into LMwMC-based thermo-reversible gel and slowly released from gel with no initial bursts. Exenatide-loaded LMwMC gel showed reduction of blood glucose level for a week in type 1 diabetic animal model. FGF 21-loaded LMwMC gel reduced glucose level to normal condition and maintained over 10days in type 2 diabetic animal model. LMwMC-based thermo-reversible and injectable hydrogel provides a strong potential to be efficient protein drug delivery system for the treatment of type 1 and type 2 diabetes.

Mild Pretreatment of Yellow Poplar Biomass using Sequential Dilute Acid and Enzymatically-generated Peracetic Acid to Enhance Cellulase Accessibility

이형래,Romas J. Kazlauskas,박태현 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.4

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140oC, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60oC, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.

Expression of Enzymatically-active Phospholipase Cγ2 in E.coli

Ozdener, Fatih,Kunapuli, Satya P.,Daniel, James L. Korean Society for Biochemistry and Molecular Biol 2002 Journal of biochemistry and molecular biology Vol.35 No.5

Phospholipase C-gamma-2 ($PLC{\gamma}2$) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to $PLC{\gamma}2$ activation, we devised a quick method for obtaining sufficient $PLC{\gamma}2$. We obtained the full-length cDNA for human $PLC{\gamma}2$ and expressed it in E. coli using the expression vector pT5T. To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons. The protein expression was detected in a Western blot analysis by both anti-$PLC{\gamma}2$ antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal. Crude lysates that were prepared from bacteria that express $PLC{\gamma}2$ were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on $PLC{\gamma}2$ that is isolated from mammalian tissue, the recombinant enzyme was $Ca^{2+}$ dependent with optimal activity at 1-10 uM $Ca^{2+}$.

Effect of Crude Microbial Enzyme Pretreatment on the Liberation of Biological Compounds and Antioxidant Activity of Red Ginseng Extract

Wei-Jie Wu,안병용 한국식품과학회 2013 Food Science and Biotechnology Vol.22 No.3

To improve the quality of red ginseng extract,the effects of crude microbial enzyme pretreatment on the liberation of biological compounds and the antioxidant activity of the extract were studied. The total ginsenoside contents in red ginseng extract pretreated with and without crude microbial enzyme were 199 and 186μg/mL, respectively. More specifically, ginsenosides with the protopanaxadiol type of aglycone moieties showed significant increases (about 10%), while the protopanaxatriol type ginsenosides were hardly changed. Ginsenosides are thermally unstable,as they may degrade during thermal extraction above 70oC,and protopanaxatriol type ginsenosides are more susceptible than protopanaxadiol type. The contents of soluble solid,reducing sugars, polyphenolic compounds, and recovery of the enzymatic-pretreated group were increased 17, 51, 10,and 17%, respectively, compared with control. Additionally,the enzymatic-pretreated red ginseng extract showed significantly higher antioxidant activity and free radical scavenging ability than control.

Enzymatic Extracts from Edible Red Algae, Porphyra tenera, and Their Antioxidant, Anti-acetylcholinesterase, and Anti-inflammatory Activities

Mahinda Senevirathne,Chang-Bum Ahn,Jae-Young Je 한국식품과학회 2010 Food Science and Biotechnology Vol.19 No.6

Enzymatic extracts from Porphyra tenera were prepared using 4 proteases (Protamex, Neutrase, Flavourzyme,and Alcalase) and 7 carbohydrases (AMG, Celluclast,Dextrozyme, Maltogenase, Termamyl, Promozyme, and Viscozyme), and biological activities of the enzymatic extracts from P. tenera were determined as antioxidant,anti-acetylcholinestrase (AChE), and anti-inflammation. The Alcalase and Maltogenase extracts showed higher 1,1-diphenyl-2-picrylhydrozyl (DPPH) scavenging activities compared to the other extracts. At the 2.5 mg/mL, 94.38%(Alacalase extracts), and 80.13% (Maltogenase extracts)scavenging capacities were observed. The Alcalase and Maltogenase extracts were also showed strong reducing power, ferrous ion chelating, and hydrogen peroxide (H2O2)scavenging capacities. In addition, 2 enzymatic extracts effectively protected hydroxyl radical-induced DNA damage. In the case of AChE inhibition, the Flavourzyme (99.32%inhibition) and Viscozyme extracts (82.68% inhibition)were observed. All enzymatic extracts showed no cytotoxicity in RAW264.7 macrophages, and all enzymatic extracts effectively inhibited lipopolysaccharides (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. These results suggest that the enzymatic extracts from P. tenera would be useful as an ingredient for functional foods.

생체 이식형 효소 기반 바이오 연료전지 연구의 최근 동향

이강진,Phan Gia Le,김문일 한국생물공학회 2021 KSBB Journal Vol.36 No.4

Enzymatic biofuel cell has been recognized as a sustainable and environmentally-friendly energy source, that is based on enzymatic catalysis to generate electric power from diverse bioresources rather than fossil energy sources. The biofuels for the enzymatic biofuel cell are generally carbohydrate relatives, such as glucose, sucrose, fructose, alcohols, organic acids, and organic salts, which are present within physiological fluids of living organisms. Thus, the enzymatic biofuel cell has been intensively investigated as a power source of implantable devices, which utilize the infinite biomoleculetype energy sources in living organisms, including plants, animals, as well as human. To highlight the research progress of implantable enzymatic biofuel cell, this review discusses the component and structure of biofuel cell, its electivity-generating mechanism, and recent research studies of implantable applications on plant, animal, and human. Finally, we address the current challenges with possible solutions, as well as future prospects of the implantable enzymatic biofuel cell.

엔테로코커스 검출을 위한 막여과법과 효소발색법의 비교

이규철(Gyu-Cheol Lee),최재원(Jae-Won Choi),최일환(Il-Hwan Choi),김윤석(Yun-Seok Kim) 한국엔터테인먼트산업학회 2011 한국엔터테인먼트산업학회논문지 Vol.5 No.3

본 연구에서는 수계에 존재하는 엔테로코커스를 검출하기 위해 막여과법과 효소발색법을 비교ㆍ검토하였다. 증류수와 멸균한 상수 원수에 엔테로코커스 표준 배양액을 농도별로 접종한 후 두 방법의 엔테로코커스 검출 결과를 비교하였다. 증류수에 접종한 경우, 막여과법에 의한 엔테로코커스 검출 회수율(84.5%)이 효소 발색법에 의한 회수율(100.9%)보다 비교적 양호하였으나 두 방법 간의 상관성(R<SUP>2</SUP>=0.928)은 매우 높았다. 멸균한 원수 시료에 엔테로코커스를 접종한 경우, 효소발색법에 의한 검출 회수율이 막여과법의 50% 수준으로 나타났다. 실제 상수 원수의 분석에 효소발색법을 적용할 수 있는지를 알아보기 위해, 20개 원수시료를 두 방법으로 분석하였다. 그 결과 효소발색법에 의해 9개 시료(45%)가, 막여과법에 의해 10개(50%) 시료가 양성으로 판정되었다. 양성율은 유사하였으나, 16S rRNA 염기서열 분석결과 효소발색법에 의해 양성으로 판정된 일부 시료가 위양성으로 판정되었다. 따라서 효소발색법을 한국의 원수 시료에 엔테로코커스 분석법으로 적용하는 것은 무리가 있을 것으로 판단되며, 시료의 특성이나 분석 목적에 따라 막여과법과 융통성 있게 적용할 수 있는 추가적인 연구가 필요할 것이다. This study evaluated the usefulness of the membrane filtration method and the enzymatic method for the enumeration of Enterococcus sp. in aquatic environment applications. Various concentration of Enterococcus were spiked in the distilled water and the sterilized source water, and the results of two methods were compared. When Enterococcus were inoculated in the distilled water, the recovery rate of the enzymatic method and the membrane filtration method were 84.5% and 100.9%, respectively, and the correlation coefficient was high (R<SUP>2</SUP>=0.928). However, in case of sterilized source water, the detection recovery rate of the enzymatic method was about 50% of the recovery rate of the membrane filtration method. In order to test the applicability of the enzymatic method for the enumeration of Enterococcus sp. in field samples, the twenty source water samples were analyzed and compared by using two methods. Nine samples (45%) were Enterococcus positive by the enzymatic method and ten (50%) were positive by the membrane filtration method. The positive ratio was similar but the false positive were observed in the positive samples by the enzymatic method. These results suggest that the enzymatic method may not be useful the enumeration of Enterococcus sp. in Korean source water and further studies for the application of the enzymatic method are needed.