pH stabilization of Escherichia coli penicillin acylase by CMC conjugation

90 kDa mol ağırlığında karboksimetilselüloz (CMC) ile konjuge olan Escherichia coli Penisilin G asilaz’ının sıcaklık ve pH’ya karşı geri dönüşümsüz inaktivasyonu çalışılmıştır. Karboksimetilselüloz periyodat oksidasyonu ile aktive edilmiş ve PGA’a, Schiff bazı oluşumu ile kovalent olarak bağlanmıştır. PGA ve modifiye olmuş PGA farklı zaman ve farklı pH değerlerinde (pH 4-9) inkübe edilmiştir. PGA’ın pH karşısındaki en yüksek stabilitesi, pH 8’de 4 kat olarak elde edilmiştir. CMC’un çapraz bağlanması sonucu PGA enziminin optimal pH ve kcat değeri değişmemiştir. Km, ve Vm değerleri ise azalmıştır. Konjuge PGA için farklı pH değerlerinde inaktivasyonun aktivasyon serbest enerjisi (ΔGi) PGA’nınkine göre daha yüksek bulunmuştur.  Anahtar Kelimeler: Enzim stabilizasyonu, Escherichia coli, karboksimetilselüloz konjugasyonu, penisilin G asilaz.The irreversible inactivation of kinetics and the stability of Escherichia coli penicillin G acylase (PGA) at various pH values conjugated by anionic polysaccharide carboxymethyl-cellulose (CMC) were studied. The use of ionic polysaccharides in enzyme technology have many potential advantages, such as water solubility, biocompatibility and non-toxicity. The stabilizing effect of ionic polysaccharides on enzyme molecules is well known by causing the cross links between the free amino group containing amino acid residues on protein molecule and strengthening  the electrostatic interactions on enzyme after conjugation. CMC was activated by periodat oxidation to its aldehyde derivative and covalently bound to PGA via Schiff's base formation. The 46% fraction of total enzyme activity and 65% fraction of total protein in PGA solution was conjugated by CMC. The amount of CMC bound protein was 33% of the initial CMC concentration. Native and CMC conjugated PGA were incubated at 40°C and different pH values between (4-9) interval for extended times. The irreversible inactivations of the native and conjugated PGA at pH values studied were obeyed to the first order inactivation kinetics. The highest pH stability of PGA was obtained at pH 8 as four fold. Cross-linking by CMC did not affect the pH profile and the kcat value of enzyme. However Km and Vm values were decreased after cross-linking. The activation free energy of inactivation (DGi) at different pH values for conjugated PGA were found to be always higher than that for native enzyme. CMC conjugation is improved the catalytic performance of enzyme by increasing the kcat/Km ratio. Keywords: Carboxymethylcellulose conjugation, enzyme stabilization, Escherichia coli, penicillin G acylase

Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions

Aim: The isolation and identification of new Bacillus sp. capable of growing under highly alkaline conditions as alkaline protease producers. Methods and Results: A Bacillus strain capable of growing under highly alkaline conditions was isolated from compost. The strain is a Gram-positive, spore-forming, motile, aerobic, catalase- and oxidase-positive, alkaliphilic bacterium and designated as GMBAE 42. Good growth of the strain was observed at pH 10. The strain was identified as Bacillus clausii according to the physiological properties, cellular fatty acid composition, G + C content of genomic DNA and 16S rRNA gene sequence analyses. The result of 16S rRNA sequence analyses placed this bacterium in a cluster with B. clausii. The G + C content of the genomic DNA of the isolate GMBAE 42 was found to be 49 mol%. The crude extracellular alkaline protease produced by the isolate showed maximal activity at pH 11.0 and 60degreesC. Conclusions: The results suggest that isolated strain GMBAE 42 is a new type of B. clausii capable of growing at pH 10.0 and produce extracellular alkaline protease very active at pH 11.0. Significance and Impact of the Study: Isolated strain could be used in commercial alkaline protease production and its enzyme can be considered as a candidate as an additive for commercial detergents

Halomonas smyrnensis sp nov., a moderately halophilic, exopolysaccharide-producing bacterium

Four Gram-negative, moderately halophilic, exopolysaccharide-producing strains, designated AAD6(T), AAD4, AAD17 and AAD21, were isolated from Camalti Saltern Area, a wildlife reserve in Sasali, Izmir province located in the Aegean Region of Turkey. The isolates grew at an optimum NaCl concentration of 10% (w/v). The major cellular fatty acids were C-16:0, C-18:1 omega 7c, C-16:1 omega 7c and C-12:0 3OH, respectively and the predominant lipoquinone was ubiquinone Q-9. The G+C content of the genomic DNA of strains AAD6(T), AAD4, AAD17 and AAD21 was 63.0, 63.3, 62.8 and 62.6 mol%, respectively. Comparative 16S rRNA gene sequence studies showed that the isolates belonged to the genus Halomonas. The DNA-DNA hybridization mean values between the representative strain AAD6(T) and the closely related species Halomonas saline DSM 5928(T), Halomonas halophila DSM 4770(T), Halomonas maura DSM 13445(T), Halomonas organivorans DSM 16226(T), Halomonas elongate DSM 2581(T), Halomonas koreensis JCM 12237(T) and Halomonas nitroreducens LMG 24185, were 40.8, 39.6, 24.2, 23.3, 12.6, 14.5 and 12.2%, respectively. Based on these data the strains represent a novel species of the genus Halomonas for which the name Halomonas smyrnensis sp. nov. is proposed. The type strain is AAD6(T) (=DSM 21644(T)=JCM 15723(T))

Proteomic Analysis of Liver Preservation Solutions Prior to Liver Transplantation

Objective: Transplantation is the preferred treatment for patients with end-stage liver diseases. However, in clinical practice, functional preservation of the liver is a major concern before the transplantation. Although various protective solutions are used (in combination with hypothermia), the functional preservation time for liver is still limited to hours. We analyzed the preservation medium to detect the proteins released from the liver during storage period. Material/Methods: Samples were collected from the pre-transplant preservation mediums of 23 liver donors. For all donors, the cases involved Donation after Brain Death (DBD). 2D-PAGE and LCMSMS methodologies were used to detect the proteins and peptides from the preservation mediums. Results: A total of 198 proteins originating from the liver were detected. Conclusion: The data provide valuable insights into biomarkers that may be used to evaluate organ injury, functional status, and suitability for transplantation. Additionally, the findings could be valuable for the development of new strategies for effective preservation of solid organs prior to transplantation

Pyrolysis kinetics and thermal characteristics of microalgae Nannochloropsis oculata and Tetraselmis sp.

In this study non-isothermal thermogravimetric analysis were used to investigate pyrolysis behavior and kinetics of microalgae Nannochloropsis oculata (NO) and Tetraselmis sp. (TS). TG/DTG experiments at different heating rates were carried out. Heating rates had slight effect on the decomposition trend, however the maximum temperature and peak of weight loss rate in the DTG curves shifted towards higher temperature with the increase in heating rate. The average activation energy and pre-exponential factor for pyrolysis of NO and TS were estimated by distributed activation energy model. The highest activation energies were observed as 152.20 and 334 kJ/mol for NO and TS, respectively, at various conversions. The pre-exponential factors for the corresponding activation energies were observed to be in the order of 10(8)-10(13) and 10(12)-10(25) s(-1) for NO and TS, respectively. Calculated kinetic parameters were used to predict devolatilization curves and results were in well agreement with experimental data. (C) 2015 Elsevier Ltd. All rights reserved

Biodiesel production from waste oils by using lipase immobilized on hydrotalcite and zeolites

In this work, hydrotalcite and four different types of zeolites were used as immobilization metarial. The size and type of the zeolite particles did not effect the amount of protein adsorbed. It was found that hydrotalcite is more efficient than zeolites studied. The amount of protein adsorbed (P-g) on hydrotalcite 13 mg/g was higher than that of zeolite as 9 mg/g. The amount of protein adsorbed on hydrotalcite was the highest at pH 8.5 and 4 degrees C. The immobilization of enzyme on hydrotalcite reached steady state after 5 h. Immobilized lipase retained 36% of the initial activity at 45 degrees C and 14% of initial activity at 55 degrees C, after the seventh cycle. Immobilized lipase on hydrotalcite was found to able to catalyse the transesterification of waste cooking oil with methanol to produce methyl esters. Lipase immobilized on zeolites did not show significant yields at the same reaction conditions. (c) 2007 Elsevier B.V. All rights reserved

Proteomic Analysis of Kidney Preservation Solutions Prior to Renal Transplantation

One of the main issues in kidney transplantation is the optimal functional preservation of the organ until its transplantation into the appropriate recipient. Despite intensive efforts, the functional preservation period remains limited to hours. During this time, as a result of cellular injury, various proteins, peptides, and other molecules are released by the organ into the preservation medium. In this study, we used proteomic techniques to analyze the protein profiles of preservation solutions in which organs had been preserved prior to their transplantation. Samples were obtained from the preservation solutions of 25 deceased donor kidneys scheduled for transplantation. The protein profiles of the solutions were analyzed using 2D gel electrophoresis/MALDI-TOF and LC-MS/MS. We identified and quantified 206 proteins and peptides belonging to 139 different groups. Of these, 111 proteins groups were belonging to kidney tissues. This study used proteomic techniques to analyze the protein profiles of organ preservation solutions. These findings will contribute to the development of improved preservation solutions to effectively protect organs for transplantation

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37 degrees C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH4)(2)SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60 degrees C; however, it is shifted to 70 degrees C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40 degrees C for 2 h at pH 10.5; only 14% activity loss was observed at 50 degrees C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0-12.2 range for 24 h at 30 degrees C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol(-1) (44.30 U mol(-1)). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30 degrees C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5phenyl-iso-xazolium-3'-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k(cat) value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. Km and kcat values were estimated at 0.655 mu M N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21 x 10(3) min(-1), respectively