We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.
Co-composting of sewage sludge and animal fat mixtures was studied in order to determine the possibility of using this technology to recycle fat-enriched wastes. A maximum fat content of 30% in fat:sludge mixtures is recommended to achieve the international sanitation requirements on compost quality and to avoid an excessive thermophilic composting time. Under these conditions a fat content reduction of 85% was achieved. Biological activity was highly dependent on the moisture content as shown by the respiratory quotient values. Moisture content is a critical control factor because of the hydrophobic nature of fats and should be maintained above 40% in the composting of fats. Biological indices of the compost obtained after 69 days of process (Maturity Grade: IV; Respiration Index: 1.1 mg O 2 consumed per g organic matter and hour) indicated a high stability and maturity degree of the material. Lipases responsible for fat hydrolysis were monitored during the composting process and a sample from the thermophilic period was characterized in terms of stability in front of pH and temperature. Optimal conditions for lipase stability were found at 38.3ºC and pH 7.97, however the maximum lipolytic activity was observed at thermophilic temperatures. Lipases from the thermophilic period were purified by anion exchange chromatography and visualised by SDS-PAGE. Two major bands were observed at molecular weights of 29 and 62 kDa. These bands could not be identified precisely by N-terminal sequence analysis.
We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)].
L-aspartate ammonia-lyase from Bacillus sp. YM55-1 (AspB, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate (Asp) into fumarate and ammonia with a high specific activity toward the substrate. AspB was expressed in Escherichia coli and partially purified by heat precipitation and saturation with ammonium sulfate reaching purification factor of 7.7 and specific activity of 334 U/mg of protein. AspB was immobilized by covalent attachment on Eupergit® C (epoxy support) and MANA-agarose (amino support), and entrapment in LentiKats® (polyvinyl alcohol) with retained activities of 24, 85 and 63 %, respectively. Diffusional limitations were only observed for the enzyme immobilized in LentiKats® and were overcome by increasing substrate concentration. Free and immobilized AspB were used for the synthesis of aspartate achieving high product concentration (≥450 mM) after 24 h of reaction. Immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, keeping over 90 % of activity and reaching over 90 % of conversion in all the cases.
The effect of secondary reactions on DHAP-dependent aldolase stereoselective synthesis yields is reported. The fuculose-1-phosphate aldolase catalyzed synthesis between DHAP and Cbz-S-alaninal has been chosen as case study. It has been demonstrated that DHAP is not only chemically degraded in the reaction medium, but also enzymatically. The last reaction has been shown to take place when type II aldolases are used as biocatalysts. In order to minimize the effect of non-desired reactions, temperature reduction has been shown to be favorable, and operation at 4 degrees C has been chosen as appropriate. On the other hand, the fed-batch addition of DHAP also increased the synthesis yields and, combined with low temperature, led to almost quantitative conversion.
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