ABSTRACT
Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.
Subject(s)
Bacterial Proteins/chemistry , Geobacillus/enzymology , Lipase/chemistry , Protein Aggregates , Bacterial Proteins/genetics , Cross-Linking Reagents/chemistry , Enzyme Stability , Geobacillus/genetics , Lipase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
A cocrystal of the antihypertensive drug chlorthalidone (CTD) with caffeine (CAF) was obtained (CTD-CAF) by the slurry method, for which a 2:1 stoichiometric ratio was found by powder and single-crystal X-ray diffraction analysis. Cocrystal CTD-CAF showed a supramolecular organization in which CAF molecules are embedded in channels of a 3D network of CTD molecules. The advantage of the cocrystal in comparison to CTD is reflected in a threefold solubility increase and in the dose/solubility ratios, which diminished from near-unit values for D0D to 0.29 for D0CC. Furthermore, dissolution experiments under non-sink conditions showed improved performance of CTD-CAF compared with pure CTD. Subsequent studies showed that CTD-CAF cocrystals transform to CTD form I where CTD precipitation inhibition could be achieved in the presence of pre-dissolved polymer HPMC 80-120 cPs, maintaining supersaturation drug concentrations for at least 180 min. Finally, dissolution experiments under sink conditions unveiled that the CTD-CAF cocrystal induced, in pH-independent manner, faster and more complete CTD dissolution when compared to commercial tablets of CTD. Due to the stability and dissolution behavior of the novel CTD-CAF cocrystal, it could be used to develop solid dosage forms using a lower CTD dose to obtain the same therapeutic response and fewer adverse effects.