scholarly journals Characterization of an Alkaline GH49 Dextranase from Marine Bacterium Arthrobacter oxydans KQ11 and Its Application in the Preparation of Isomalto-Oligosaccharide

Marine Drugs ◽  
2019 ◽  
Vol 17 (8) ◽  
pp. 479 ◽  
Author(s):  
Hongfei Liu ◽  
Wei Ren ◽  
Mingsheng Ly ◽  
Haifeng Li ◽  
Shujun Wang
Keyword(s):  
Escherichia Coli ◽  
Marine Bacteria ◽  
Recombinant Expression ◽  
Bifidobacterium Longum ◽  
Lactobacillus Rhamnosus ◽  
Km Value ◽  
Tlc Analysis ◽  
Layer Chromatography ◽  
Arthrobacter Oxydans

A GH49 dextranase gene DexKQ was cloned from marine bacteria Arthrobacter oxydans KQ11. It was recombinantly expressed using an Escherichia coli system. Recombinant DexKQ dextranase of 66 kDa exhibited the highest catalytic activity at pH 9.0 and 55 °C. kcat/Km of recombinant DexKQ at the optimum condition reached 3.03 s−1 μM−1, which was six times that of commercial dextranase (0.5 s−1 μM−1). DexKQ possessed a Km value of 67.99 µM against dextran T70 substrate with 70 kDa molecular weight. Thin-layer chromatography (TLC) analysis showed that main hydrolysis end products were isomalto-oligosaccharide (IMO) including isomaltotetraose, isomaltopantose, and isomaltohexaose. When compared with glucose, IMO could significantly improve growth of Bifidobacterium longum and Lactobacillus rhamnosus and inhibit growth of Escherichia coli and Staphylococcus aureus. This is the first report of dextranase from marine bacteria concerning recombinant expression and application in isomalto-oligosaccharide preparation.

scholarly journals Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adaptedLactobacillus rhamnosusGG

2017 ◽  
Vol 73 (6) ◽  
pp. 321-327 ◽  
Author(s):  
Arjun K. Mishra ◽  
Abhin Kumar Megta ◽  
Airi Palva ◽  
Ingemar von Ossowski ◽  
Vengadesan Krishnan
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Molecular Mechanisms ◽  
Recombinant Expression ◽  
Lactobacillus Rhamnosus ◽  
X Ray Diffraction ◽  
X Ray ◽  
Pilin Subunit ◽  
Sad Phasing

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensalLactobacillus rhamnosusGG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria,L. rhamnosusGG SpaE for crystallization was produced by recombinant expression inEscherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.

scholarly journals Ekstraksi, Karakterisasi dan Uji Aktivitas Antioksidan Astaxanthin dari Produk Fermentasi Udang (Cincalok)

2021 ◽  
Vol 24 (3) ◽  
pp. 311-322
Author(s):  
Mauludia Mauludia ◽  
Thamrin Usman ◽  
Winda Rahmalia ◽  
Dwi Imam Prayitno ◽  
Siti Nani Nurbaeti
Keyword(s):  
Antioxidant Activity ◽  
High Pressure ◽  
Liquid Chromatography ◽  
High Pressure Liquid Chromatography ◽  
Aquatic Organisms ◽  
Dpph Method ◽  
Wet Weight ◽  
Tlc Analysis ◽  
Layer Chromatography

Shrimp is one of the aquatic organisms that contain several active compounds, including astaxanthin. Cincalok is one of the fermented shrimp products containing astaxanthin. This study aims to determine the characteristics of astaxanthin extract from cincalok and its antioxidant activity. Extraction of astaxanthin from cincalok was carried out using the reflux method with acetone : cyclohexane (20:80 v/v) as a solvent. The identification and characterization of astaxanthin was carried out using thin-layer chromatography (TLC), UV-Vis spectrophotometry, and High-Pressure Liquid Chromatography (HPLC). Meanwhile, the antioxidant activity test was carried out using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method in one serial concentration (5; 15; 25 ppm). The results of TLC analysis showed that astaxanthin in cincalok extract has Rf value (0.32). The analysis using a UV-Vis spectrophotometer produced a spectrum with a maximum wavelength of 477 nm, which corresponds to the maximum wavelength of standard astaxanthin. The yield of astaxanthin extract from cincalok in this study was 1.47 mg/100 g wet weight. The chromatogram from the results of UHPLC analysis showed that the retention time of cincalok astaxanthin extract was 6.27 minutes with a purity of 18.03%. The antioxidant activity of cincalok astaxanthin extract was 568.32 ppm. Udang merupakan salah satu organisme air yang mengandung banyak senyawa aktif, termasuk astaxanthin. Cincalok merupakan salah satu produk hasil fermentasi udang yang mengandung astaxanthin. Penelitian ini bertujuan untuk mengetahui karakteristik ekstrak astaxanthin dari cincalok dan aktivitas antioksidannya. Ekstraksi astaxanthin dari cincalok menggunakan metode refluks dengan pelarut aseton:sikloheksan (20:80 v/v). Identifikasi dan karakterisasi astaxanthin dilakukan dengan menggunakan kromatografi lapis tipis (KLT), spektrofotometri UV-Vis, dan High Pressure Liquid Chromatography (HPLC). Sedangkan uji aktivitas antioksidan dilakukan menggunakan metode 1,1-difenil-2-pikrilhidrazil (DPPH) dengan memvariasikan konsentrasi larutan uji, yaitu 5; 15; 25 ppm. Hasil dari penelitian ini melaporkan astaxanthin pada ekstrak cincalok menunjukkan nilai Rf 0,32 pada kromatografi lapis tipis (KLT). Hasil analisis menggunakan spektrofotometer UV-Vis menghasilkan spektra dengan panjang gelombang maksimum 477 nm, yang sesuai dengan panjang gelombang maksimum astaxanthin standar. Randemen ekstrak astaxanthin dari cincalok pada penelitian ini adalah 1,47 mg/100 g berat basah. Kromatogram dari hasil analisis UHPLC menunjukkan waktu retensi ekstrak astaxanthin cincalok yaitu selama 6,27 menit dengan kemurnian sebesar 18,03%. Aktivitas antioksidan dari ekstrak astaxanthin cincalok diperoleh sebesar 568,32 ppm.  

Recombinant expression, purification and characterization of antimicrobial peptide ORBK in Escherichia coli

2014 ◽  
Vol 95 ◽  
pp. 182-187 ◽  
Author(s):  
Yan Li ◽  
Jiarong Wang ◽  
Jing Yang ◽  
Chanjuan Wan ◽  
Xiaoming Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Recombinant Expression ◽  
Purification And Characterization

scholarly journals Identification and Characterization of an Archaeon-Specific Riboflavin Kinase

2008 ◽  
Vol 190 (7) ◽  
pp. 2615-2618 ◽  
Author(s):  
Zahra Mashhadi ◽  
Hong Zhang ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Recombinant Expression ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Gene Recombinant ◽  
Identification And Characterization

ABSTRACT The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

scholarly journals The Attractant Bioactivity Test of Semi-Polar Fraction of the Datuan Stem Bark (Ficus vasculosa Wall. Ex Miq) against Warehouse Pest (Sitophilus oryzae L.)

2021 ◽  
Author(s):  
Syaiful Bahri ◽  
Yuli Ambarwati ◽  
Lina Marlina ◽  
Vera Fitriani ◽  
Sutopo Hadi
Keyword(s):  
Mass Spectrometry ◽  
Absorption Band ◽  
Sitophilus Oryzae ◽  
Stem Bark ◽  
Separation And Purification ◽  
Eluent Ethyl Acetate ◽  
Isolated Compound ◽  
Tlc Analysis ◽  
Layer Chromatography

Bioactive isolation was performed on the stem bark of Datuan (Ficus vasculosa Wall. Ex Miq), and extraction was carried out via the maceration method using acetone as a solvent. Furthermore, an attractant bioactivity test was conducted on acetone extract, A-G fraction, and composition of the isolates. The separation and purification via column chromatography produced a D8.3.5.7 fraction in the form of needle crystal of about 50 mg, at a melting point of 136°C–138.7°C. Thin-layer chromatography (TLC) analysis showed a single spot at an Rf value of 0.57 (n-hexane eluent: ethyl acetate 7:3), 0.36 (DCM eluent), and 0.24 (CHCl3 eluent). The isolated compounds were identified using infrared and UV–Vis spectrophotometry, as well as mass spectrometry. The characterization of the infrared spectrum of the isolated compound showed a strong OH goo band at 3461 cm-1 region and the absorption band at 2936.25 cm-1 exhibited a stretch of CH alkanes. These two bands are supported by the vibration at 1378.47 and 1462.55 cm-1 for CH absorption of methyl and methylene. The absorption band in the 1622 cm-1 region showed a stretch of conjugated C=C double bond, which is supported by absorption at 918.96 and 966.22 cm-1 as C–H alkene. The UV–Vis spectrophotometry showed absorption at λmax 263.97 nm A = 0.483, which was the result of electronic transition π → π*, and at λ 331.0 nm A = 0.274, which was an electronic result of n → π*. Meanwhile, identification via mass spectrometry that produces isolate has a molecular weight of 414.1 m/e with the formula C29H50O. Therefore, the bioactivity test results on compound D8.3.5.7 had an attractant activity of 71.67% against warehouse pests (Sitophilus oryzae L.) and an interest index of 0.63.

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