scholarly journals Electrophoretic and zymographic techniques for production monitoring of two lipase forms from Candida antarctica DSM 70725

2012 ◽  
Vol 66 (2) ◽  
pp. 201-206
Author(s):  
Aleksandra Dimitrijevic ◽  
Dusan Velickovic ◽  
Ratko Jankov ◽  
Nenad Milosavic
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Fermentation Broth ◽  
Candida Antarctica ◽  
Simple Method ◽  
Wide Range ◽  
Work Production ◽  
Lipase A ◽  
Dimeric Form ◽  
Hydrolysis Of

Yeast Candida antarctica produces two lipase forms, which are widely used as catalysts in variety of organic reactions, many of which are applied on a large scale. In this work, production of two forms of lipase from C. antarctica DSM 70725 (CAL A and CAL B) was monitored during seven days of cultivation in the optimal medium using different electrophoretic and zymographic techniques. According to electrophoresis after silver staining, C. antarctica lipase A (molecular mass 45 kDa) was produced starting from the second day of cultivation. C. antarctica lipase B (CAL B) was also produced starting from the second day, but protein was present in the fermentation broth predominantly as dimer (molecular weight 66 kDa), while presence of monomeric form of CAL B (molecular weight of 33 kDa) was observed starting from the fourth day of cultivation. Both types of zymograms (based on hydrolysis and synthesis reactions) were used for detection of lipase activity in the fermentation broth. C. antarctica lipase A showed activity only in hydrolytic zymogram, when ?-naphtyl butyrate was used as substrate. In the same zymogram, with ?-naphtyl acetate as substrate no CAL A activity was detected. Similarly, CAL A showed no activity in synthesis based zymograms towards oleic acid and octanol as substrates, indicating that CAL A is not active towards very short or long-chain substrates. As opposite of CAL A, both monomeric and dimeric form of CAL B were detected in the all zymograms, suggesting that CAL B is active towards wide range of substrates, regardless to the chain length. Thus, zymogram based on hydrolysis of ?-naphtyl butyrate represents a simple method for monitoring the production of two forms of lipase from C. antarctica, that greatly differ in their characteristics.

scholarly journals A simple method for the production of low molecular weight hyaluronan by in situ degradation in fermentation broth

e-Polymers ◽  
2019 ◽  
Vol 19 (1) ◽  
pp. 477-481
Author(s):  
Jianfeng Mei ◽  
Zhihong Dong ◽  
Yu Yi ◽  
Yanlu Zhang ◽  
Guoqing Ying
Keyword(s):  
Molecular Weight ◽  
Enzymatic Degradation ◽  
Fermentation Broth ◽  
Low Molecular Weight ◽  
Streptococcus Zooepidemicus ◽  
Medium Ph ◽  
Simple Method ◽  
Suitable Temperature ◽  
In Situ Degradation

AbstractFermentation of hyaluronan (HA) by Streptococcus zooepidemicus was carried out in a 10-L fermentor. When the medium pH was controlled at 7.0 and the temperature was maintained at 38°C for 12 h followed by 35°C for 8 h, the yield of HA was 4.83 g/L with a molecular weight of 1,890 kDa. After the cells were removed by centrifugation from the fermentation broth, HA was slowly degraded to low molecular weight HA by hyaluronidase at a suitable temperature without a decrease in HA concentration. If the time and temperature for enzymatic degradation were controlled, the desired low molecular weight HA could be obtained by in situ degradation in the fermentation broth. The method does not require the addition of exogenous hyaluronidase, and is a simple way to produce low molecular weight HA.

scholarly journals Facile Molecular Weight Determination of Polymer Brushes Grafted from One-Dimensional Diffraction Grating by SI-ATRP Using Reflective Laser System

Polymers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 4270
Author(s):  
Jem-Kun Chen ◽  
Feng-Ping Lin ◽  
Chi-Jung Chang ◽  
Chien-Hsing Lu ◽  
Chih-Feng Huang
Keyword(s):  
Molecular Weight ◽  
Polymer Brushes ◽  
Diffraction Effect ◽  
Molecular Weight Determination ◽  
Diffraction Intensity ◽  
Simple Method ◽  
Grafting Polymerization ◽  
One Dimensional ◽  
Wide Range

Gelatin was immobilized selectively on the amide groups-modified bottom of a trench array of a photoresist template with 2 μm resolution by the ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide reaction. The gelatin-immobilized line array was brominated to generate a macroinitiator for atom transfer radical polymerization. Poly(methacrylic acid) (PMAA) brushes were grafted from the macroinitiator layer as line arrays of one-dimensional diffraction gratings (DGs) for various grafting polymerization times. A laser beam system was employed to analyze the optical feature with a characteristic diffraction effect of the PMAA DGs at a 45° incident angle along the transverse magnetic and transverse electric polarization. The growth of the PMAA brush lines increased both their heights and widths, leading to a change in the reflective diffraction intensity. The PMAA brushes under various grafting polymerization times were cleaved from the substrate by digestion of gelatin with trypsin, and their molecular weights were obtained by gel permeation chromatography. The change degree of the diffraction intensity varied linearly with the molecular weight of the PMAA brushes over a wide range, from 135 to 1475 kDa, with high correlation coefficients. Molecular weight determination of polymer brushes using the reflective diffraction intensity provides a simple method to monitor their growth in real time without polymer brush cleavage.

Human and Bovine Plasminogen-Free Thrombin, Purified by Means of Gel Filtration and Ion Exchange Chromatography

1966 ◽  
Vol 15 (03/04) ◽  
pp. 501-510 ◽  
Author(s):  
W Berg ◽  
K Korsan-Bengtsen ◽  
J Ygge
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Large Scale ◽  
Gel Filtration ◽  
Trisodium Citrate ◽  
Exchange Chromatography ◽  
Simple Method ◽  
Commercial Preparation ◽  
Usual Manner ◽  
Low Ionic Strength

SummaryA simple method for preparation of plasminogen-free human and bovine thrombin is described.Crude thrombin was prepared in the usual manner from oxalated plasma by means of adsorption on BaSO4, elution with trisodium citrate and activating the eluate from BaSO4 with tissue thromboplastin.This crude thrombin was purified by means of gel-filtration and chromatography on CM-Sephadex A-50.The gel-filtration was performed on three types of Sephadex, G-75, G-50, and G--25. By means of Sephadex G-75 the thrombin was well separated from the main part of inert protein and this type of Sephadex was used for the purification in large-scale. Separation of thrombin from protein of higher molecular weight was also obtained with Sephadex G-50 but not with Sephadex G-25 indicating a molecular weight of thrombin between 4000 and 10,000.The importance of using an elution buffer of sufficient high ionic strength for gel-filtration is shown. A great deal of the thrombin was adsorbed to the Sephadex if the gel-filtration was performed at a too low ionic strength.The final preparation contained 30,000 NIH units of thrombin per mg tyrosin and no detectable plasminogen.The commercial preparation “Topostasine” was also purified in the same manner, but the plasminogen content in “Topostasine” was high and could not be completely separated from thrombin.

Biopolymer Production by the Yeast Rhodotorula glutinis

2012 ◽  
Vol 550-553 ◽  
pp. 1048-1051
Author(s):  
Jong Hoon Kim ◽  
Eui Yong Kim ◽  
Khim Hoong Chu
Keyword(s):  
Molecular Weight ◽  
Stationary Phase ◽  
Apparent Viscosity ◽  
Fermentation Broth ◽  
Rhodotorula Glutinis ◽  
Exponential Phase ◽  
Partial Hydrolysis ◽  
Fermentation Time ◽  
Excess Carbon ◽  
Hydrolysis Of

In this work, exopolysaccharide (EPS) production by the yeast Rhodotorula glutinis was investigated. The results suggest that the synthesis of EPS in batch fermentations was enhanced by using a low C/N ratio to stimulate fast cell growth during the exponential phase and by adding glucose to stationary phase cultures to provide excess carbon for EPS formation. The apparent viscosity and molecular weight of EPS were found to decrease with increasing fermentation time due to the partial hydrolysis of EPS by the acidic fermentation broth (pH 1.8). Maintaining stationary phase cultures at pH 4 was found to be effective in minimizing acid hydrolysis and producing EPS with high molecular weight and apparent viscosity.

Purification and properties of a phenol carboxylic acid acyl esterase from Aspergillus flavus

1971 ◽  
Vol 17 (11) ◽  
pp. 1455-1463 ◽  
Author(s):  
J. J. Child ◽  
T. Oka ◽  
F. J. Simpson ◽  
H. G. Krishnamurty
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Carboxylic Acid ◽  
Aspergillus Flavus ◽  
Protocatechuic Acid ◽  
Hydroxybenzoic Acid ◽  
Purification And Properties ◽  
Wide Range ◽  
Hydrolysis Of ◽  
Active Preparation

Aspergillus flavus produces an extracellular esterase that hydrolyses phenolic carboxylic acid acyl esters. An assay based upon the measurement of the rate of release of phloroglucinol on hydrolysis of the ester of phloroglucinol and protocatechuic acid is described. The most active preparation hydrolyzed 30.8 μmoles of substrate per minute per milligram of protein and was active against a wide range of esters of meta and para hydroxybenzoic acid derivatives.The enzyme was isolated as a homogeneous protein, as judged by ultracentrifugation and by electrophoresis and had an isoelectric point of pH 4.45. The molecular weight was determined as 166 000. The enzyme is a glycoprotein containing 42.8% carbohydrate and the amino acid composition is described.

scholarly journals Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor

2021 ◽  
Author(s):  
Lei Cheng ◽  
Kangming Tian ◽  
Nokuthula Peace Mchunu ◽  
Dandan Niu ◽  
Meng Zhang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Scale Production ◽  
Current Investigation ◽  
Large Scale Production ◽  
Wide Range ◽  
Maximal Yield

Abstract Objectives To heterologously express peanut ( Arachis ipaensis ) Bowman-Birk inhibitor (BBI) and characterize its properties. Results A putative BBI gene from peanut was overexpressed in Pichia pastoris with the maximal yield of 11.1 mg/L in a 250-mL shaking flask fermentation. The recombinant peanut BBI (rPBBI) was purified and its molecular weight was estimated to be 9 kDa. Purified rPBBI showed 5223.6 TIA/mg inhibitory activity toward trypsin. It retained more than 95% of its inhibitory activity over wide range of temperatures (40 to 90 o C) and pH (2.0 to 10.0) after incubation for 60 min. When presence of 100 mM dithiothreitol, rPBBI lost more than 80% inhibitory activity in 30 min. Conclusion The current investigation expressed a peanut BBI with stable inhibitory activity against trypsin and exposed the potential of heterologous overexpression approach in large scale production of BBI.

scholarly journals Purification and Characterization of Nitphym, a Robust Thermostable Nitrilase From Paraburkholderia phymatum

2021 ◽  
Vol 9 ◽  
Author(s):  
Thomas Bessonnet ◽  
Aline Mariage ◽  
Jean-Louis Petit ◽  
Virginie Pellouin ◽  
Adrien Debard ◽  
...  
Keyword(s):  
Large Scale ◽  
Industrial Applications ◽  
Purification And Characterization ◽  
Thermostable Enzymes ◽  
Major Interest ◽  
Wide Range ◽  
Ph Range ◽  
Thermophilic Organisms ◽  
Hydrolysis Of

Despite the success of some nitrilases in industrial applications, there is a constant demand to broaden the catalog of these hydrolases, especially robust ones with high operational stability. By using the criteria of thermoresistance to screen a collection of candidate enzymes heterologously expressed in Escherichia coli, the enzyme Nitphym from the mesophilic organism Paraburkholderia phymatum was selected and further characterized. Its quick and efficient purification by heat treatment is of major interest for large-scale applications. The purified nitrilase displayed a high thermostability with 90% of remaining activity after 2 days at 30°C and a half-life of 18 h at 60°C, together with a broad pH range of 5.5–8.5. Its high resistance to various miscible cosolvents and tolerance to high substrate loadings enabled the quantitative conversion of 65.5 g⋅L–1 of 3-phenylpropionitrile into 3-phenylpropionic acid at 50°C in 8 h at low enzyme loadings of 0.5 g⋅L–1, with an isolated yield of 90%. This study highlights that thermophilic organisms are not the only source of industrially relevant thermostable enzymes and extends the scope of efficient nitrilases for the hydrolysis of a wide range of nitriles, especially trans-cinnamonitrile, terephthalonitrile, cyanopyridines, and 3-phenylpropionitrile.

Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features

2010 ◽  
Vol 8 (4) ◽  
pp. 886 ◽  
Author(s):  
Arto Liljeblad ◽  
Pauli Kallio ◽  
Marita Vainio ◽  
Jarmo Niemi ◽  
Liisa T. Kanerva
Keyword(s):  
Candida Antarctica ◽  
Amide Bonds ◽  
Lipase A ◽  
Hydrolysis Of

Electron microscopy study of reactively sputter-deposited Ti/N films on silicon

1992 ◽  
Vol 50 (2) ◽  
pp. 1362-1363
Author(s):  
V. C. Kannan ◽  
A. K. Singh ◽  
R. B. Irwin ◽  
S. Chittipeddi ◽  
F. D. Nkansah ◽  
...  
Keyword(s):  
Integrated Circuits ◽  
Large Scale ◽  
Hexagonal Phase ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Process Conditions ◽  
Tin Films ◽  
Wide Range ◽  
Fcc Phase ◽  
Circuits Vlsi

Titanium nitride (TiN) films have historically been used as diffusion barrier between silicon and aluminum, as an adhesion layer for tungsten deposition and as an interconnect material etc. Recently, the role of TiN films as contact barriers in very large scale silicon integrated circuits (VLSI) has been extensively studied. TiN films have resistivities on the order of 20μ Ω-cm which is much lower than that of titanium (nearly 66μ Ω-cm). Deposited TiN films show resistivities which vary from 20 to 100μ Ω-cm depending upon the type of deposition and process conditions. TiNx is known to have a NaCl type crystal structure for a wide range of compositions. Change in color from metallic luster to gold reflects the stabilization of the TiNx (FCC) phase over the close packed Ti(N) hexagonal phase. It was found that TiN (1:1) ideal composition with the FCC (NaCl-type) structure gives the best electrical property.

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