RESUMEN
Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the active-site entrance by longer loops originating from Fusarium solani cutinase and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the interaction surface for the substrate. The resulting loop hybrids showed enantioselectivities inverted toward the desired enantiomer of IPG. The acetylxylan esterase-derived variant showed an inversion in enantiomeric excess (ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was further improved by directed evolution to an ee of +57.4%.
Asunto(s)
Bacillus subtilis/enzimología , Lipasa/química , Lipasa/metabolismo , Ingeniería de Proteínas/métodos , Acetilesterasa/química , Acetilesterasa/genética , Secuencia de Aminoácidos , Sitios de Unión , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Evolución Molecular Dirigida , Lipasa/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
BACKGROUND: Since its launch, the Internet has developed into a mass medium with 1.6 billion people using it worldwide. Due to anonymity, its wide reach and the infinite stream of information from the Internet, almost anything can be found on it. This includes medicines that can normally only be acquired by way of a doctor's prescription. CASE DESCRIPTION: A 27-year-old man made a suicide attempt using psychoactive drugs he got from an illegal website in India. This caused him to develop status epilepticus, rhabdomyolysis, renal insufficiency and pulmonary oedema for which he had to be admitted to intensive care. The patient was treated with medicine, cardioversion, ventilation and haemofiltration and recovered. He was referred to a psychiatric centre. CONCLUSION: The number of illegal online pharmaceutical websites on the Internet has increased drastically in the last decade. These websites sell medicines without prescription to consumers and the traditional doctor/patient consultation does not therefore take place. Many medicines that are delivered contain the wrong concentration or the wrong active ingredient and are often contaminated with other substances. The ease with which this life-threatening medicine can be ordered online without a doctor's supervision is a possible risk to public health.