Proizvodnja lipaze iz Pseudozyma aphidis i utvrđivanje aktivnosti i stabilnosti lipaze u polarnim organskim rastvaračima

The production of lipase from Pseudozyma aphidis (DSM 70725) was determined in six different media. The highest lipase production was observed in a medium with glucose as the sole carbon source, and yeast extract and sodium nitrate as the nitrogen sources. The time course studies of growth and lipase production in the optimal medium revealed that the highest lipase production was achieved at the end of the log phase of growth, reaching the value of 35.0 U cm(-3) in the fifth day of cultivation. The effects of various polar, water-miscible, organic solvents on the activity and stability of the crude lipase produced by P. aphidis were evaluated. The hydrolytic activity of the crude lipase towards p-nitrophenyl palmitate (p-NPP) in aqueous media and in organic solvents was determined, using the same spectrophotometric assay in both the aqueous and organic media. The crude lipase preparation exhibited activity towards p-NPP only in acetone and acetonitrile, while the lipase was stable only in acetone, with 23 % residual activity after 24 h of incubation. These results suggested that lipase from P. aphidis can be used as a biocatalyst for potential applications in such organic solvents.Proizvodnja lipaze iz Pseudozyma aphidis utvrđena je u šest različitih medijuma. Najviša proizvodnja uočena je u medijumu gde je glukoza bila izvor ugljenika, a ekstrakt kvasca i natrijum-nitrat izvori azota. Praćenjem dinamike rasta i proizvodnje lipaze u optimalnom medijumu, uočeno je da se najviša proizvodnja lipaze dostiže pred kraj logaritamske faze rasta, i dostiže vrednost od 35 U cm-3 u petom danu kultivacije, što je četri puta veća proizvodnja od one do sada prijavljene u literaturi. Utvrđen je efekat različitih polarnih organskih rastvarača, mešljivih sa vodom, na aktivnost i stabilnost lipaze iz P. aphidis. Hidrolitička aktivnost lipaze prema para-nitrofenil-palmitatu (p-NPP-u) u vo- denoj sredini i organskim rastvaračima utvrđena je upotrebom istog spektrofotometrijskog testa. Pokazano je da lipaza ima aktivnost prema p-NPP-u samo u acetonu i acetonitrilu, dok je enzim stabilan jedino u acetonu i zadržava 23% aktivnosti nakon 24 časa inkubacije. Dobijeni rezultati ukazuju da lipaza iz P. aphidis može biti korišćena kao biokatalizator za potencijalne primene u acetonu kao medijumu

Proizvodnja lipaze iz Pseudozyma aphidis i utvrđivanje aktivnosti i stabilnosti lipaze u polarnim organskim rastvaračima

The production of lipase from Pseudozyma aphidis (DSM 70725) was determined in six different media. The highest lipase production was observed in a medium with glucose as the sole carbon source, and yeast extract and sodium nitrate as the nitrogen sources. The time course studies of growth and lipase production in the optimal medium revealed that the highest lipase production was achieved at the end of the log phase of growth, reaching the value of 35.0 U cm-3 in the fifth day of cultivation. The effects of various polar, water-miscible, organic solvents on the activity and stability of the crude lipase produced by P. aphidis were evaluated. The hydrolytic activity of the crude lipase towards p-nitrophenyl palmitate (p-NPP) in aqueous media and in organic solvents was determined, using the same spectrophotometric assay in both the aqueous and organic media. The crude lipase preparation exhibited activity towards p-NPP only in acetone and acetonitrile, while the lipase was stable only in acetone, with 23% residual activity after 24 h of incubation. These results suggested that lipase from P. aphidis can be used as a biocatalyst for potential applications in such organic solvents.Proizvodnja lipaze iz Pseudozyma aphidis utvrđena je u šest različitih medijuma. Najviša proizvodnja uočena je u medijumu gde je glukoza bila izvor ugljenika, a ekstrakt kvasca i natrijum-nitrat izvori azota. Praćenjem dinamike rasta i proizvodnje lipaze u optimalnom medijumu, uočeno je da se najviša proizvodnja lipaze dostiže pred kraj logaritamske faze rasta, i dostiže vrednost od 35 U cm-3 u petom danu kultivacije, što je četri puta veća proizvodnja od one do sada prijavljene u literaturi. Utvrđen je efekat različitih polarnih organskih rastvarača, mešljivih sa vodom, na aktivnost i stabilnost lipaze iz P. aphidis. Hidrolitička aktivnost lipaze prema para-nitrofenil-palmitatu (p-NPP-u) u vo- denoj sredini i organskim rastvaračima utvrđena je upotrebom istog spektrofotometrijskog testa. Pokazano je da lipaza ima aktivnost prema p-NPP-u samo u acetonu i acetonitrilu, dok je enzim stabilan jedino u acetonu i zadržava 23% aktivnosti nakon 24 časa inkubacije. Dobijeni rezultati ukazuju da lipaza iz P. aphidis može biti korišćena kao biokatalizator za potencijalne primene u acetonu kao medijumu

Production of lipase from Pseudozyma aphidis and determination of the activity and stability of the crude lipase preparation in polar organic solvents

The production of lipase from Pseudozyma aphidis (DSM 70725) was determined in six different media. The highest lipase production was ob­served in a medium with glucose as the sole carbon source, and yeast extract and sodium nitrate as the nitrogen sources. The time course studies of growth and lipase production in the optimal medium revealed that the highest lipase production was achieved at the end of the log phase of growth, reaching the va­lue of 35.0 U cm-3 in the fifth day of cultivation. The effects of various polar, water-miscible, organic solvents on the activity and stability of the crude lipase produced by P. aphidis were evaluated. The hydrolytic activity of the crude li­pase towards p-nitrophenyl palmitate (p-NPP) in aqueous media and in organic solvents was determined, using the same spectrophotometric assay in both the aqueous and organic media. The crude lipase preparation exhibited activity to­wards p-NPP only in acetone and acetonitrile, while the lipase was stable only in acetone, with 23 % residual activity after 24 h of incubation. These results suggested that lipase from P. aphidis can be used as a biocatalyst for potential applications in such organic solvents

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

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 α-naphtyl butyrate was used as substrate. In the same zymogram, with α-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 α-naphtyl butyrate represents a simple method for monitoring the production of two forms of lipase from C. antarctica, that greatly differ in their characteristics

A highly efficient diastereoselective synthesis of alpha-isosalicin by maltase from Saccharomyces cerevisiae

In this report, alpha-isosalicin, a potent anticoagulant and skin whitening agent, was synthesized by a highly efficient chemoselective and diastereoselective reaction, catalyzed by maltase from bakers' yeast (Saccharomyces cerevisiae). The highest yield of this one-step transglucosylation reaction was achieved with 50 mM of salicyl alcohol as a glucose acceptor. The key reaction factors were optimized using response surface methodology (RSM) with an enzyme concentration of 10 U/mL. The optimum temperature of the reaction was determined as 36.5 degrees C, the optimal maltose concentration was 40% (w/v), the optimal pH was 6.5, and the optimal reaction time was 16 h. Under these conditions 75% of alpha-isosalicin was obtained, with a yield of 10 g/L, and no by product formation was observed

Application of alginates in cell and enzyme immobilization

Enzymes are a particularly versatile class of catalysts that perform and regulate processes in living matter. Enzymatic regio-, chemo- and enantioselectivity were used in industry and in organic synthesis. The common perception is however, that enzymes are sensitive, unstable and have to be used in water, features that are not ideal for a catalyst and undesirable in most syntheses. In many cases a way to avoid at least part of these complaints is to immobilize enzymes. There are several immobilization techniques, and the best means of avoiding any negative influence on the structure of an enzyme is to encapsulate it. Due to its ability to form gel with multivalent cations under relatively mild conditions, alginates are very important in cell and enzyme encapsulation. Entrapment within insoluble alginate gel is recognized as a rapid, nontoxic, inexpensive and versatile method for immobilization of enzymes as well as cells. The resultant gel is biochemically inert and mechanically stable with interstitial spaces that are suitable for cell immobilization

One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite

Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10 mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83 U/mg specific activity. Silver staining after SDS-PAGE revealed the presence a single band of 45 kDa. The enzyme exhibited a temperature optimum of 60 degrees C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn2+, Ca2+, Mg2+, Cu2+, Mn2+) and stimulated by 50 mM Fe2+. Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect. (C) 2011 Elsevier Ltd. All rights reserved

Initial and residual efficacy of insecticides on different surfaces against rice weevil Sitophilus oryzae (L.)

The aim of the study was to investigate how various types of storage facilities with, e.g., concrete, metal, and plywood surfaces interfere with the activity of different insecticide formulations used for rice weevil Sitophilus oryzae (L.) control: malathion (EC), pirimiphos-methyl (EC), and lambda-cyhalothrin (CS and WP). Initial and residual efficacy were determined in the laboratory. Knockdown data for the initial effects were processed by probit analysis and presented as knockdown time (KDT) parameters with kdt-p lines. Delayed effects were shown as knockdown efficacy (%) determined after 24 h of weevils’ contact with 7-, 14-, 30-, 60-, 90-, 120-, 150-, and 180-day-old deposits on each surface. Malathion (EC) and pirimiphos-methyl (EC) showed the highest initial knockdown efficacy on metal, while it was 3.6 (3.4)- and 4.4 (3.3)-fold lower on concrete and plywood, respectively. Lambda-cyhalothrin (CS and WP) showed the highest initial efficacy on concrete, and slightly lower (1.3 and 2.4) fold on metal and plywood, respectively. Both formulations of lambda-cyhalothrin and malathion on metal, as well as pirimiphos-methyl on plywood were 100 % efficient against S. oryzae 180 days after the treatment. Delayed efficacy of both formulations of lambda-cyhalothrin decreased on plywood after 120 days, and after 180 days the efficacy was 55 %. All insecticides, except lambda-cyhalothrin (CS), expressed low knockdown efficacy on concrete, while the deposit of lambda-cyhalothrin (CS) on concrete was 100 % efficient during 90 days, and after 120, 150, and 180 days the efficacy was 83, 65, and 17 %, respectively

Holocaust Learning Handbook

"Priručnik za učenje o Holokaustu" je autorsko delo 19 profesora iz Srbije koji su 2008. godine pohađali međunarodnu školu učenja o Holokaustu u Jad Vašemu u Jerusalimu. Sastavljanjem ovakvog praktikuma želeli su da podele sa svojim kolegama u Srbiji ono što su naučili tokom studijske posete ovoj instituciji. Obrađeni su različiti pedagoško-metodički problemi sa kojima se suočavaju profesori koji mladoj generaciji treba da objasne šta je Holokaust."Priručnik za učenje o Holokaustu" (Holocaust Learning Handbook) is a work of 19 professors from Serbia who attended the International Holocaust Learning School in Yad Vashem in Jerusalem in 2008. By creating such a practicum, they wanted to share with their colleagues in Serbia what they learned during their study visit to that institution. Various pedagogical and methodological problems that professors who need to explain to the young generation about the Holocaust are addressed.Fotografija na prednjoj korici: Dolina zajednica nastalih u Holokaustu, Jad Vašem, Jerusalim, Izrael, detalj. Fotografija na zadnjoj korici: Jedan od spomenika žrtvama Holokausta u Jad Vašemu, Jerusalim, Izrael