Approaches for the enzymatic synthesis of alkyl hydroxycinnamates and applications thereof.

Alkyl hydroxycinnamates (AHs) is a group of molecules of biotechnological interest due to their cosmetic, food, and pharmaceutical applications. Among their most interesting uses are as UV protectants, skin depigmentation agents, and antioxidant ingredients which are often claimed for their antitumoral potential. Nowadays, many sustainable enzymatic approaches using low-cost starting materials are available and interesting immobilization techniques are helping to increase the reuse of the biocatalysts, allowing the intensification of the processes and increasing AHs accessibility. Here a convenient summary of AHs most interesting biological activities and possible applications is presented. A deeper analysis of the art state to obtain AHs, focusing on most employed enzymatic synthesis approaches, their sustainability, acyl donors relevance, and most interesting enzyme immobilization strategies is provided.Key points• Most interesting alkyl hydroxycinnamates applications are summarized.• Enzymatic approaches to obtain alkyl hydroxycinnamates are critically discussed.• Outlook of enzyme immobilization strategies to attain alkyl hydroxycinnamates.

Biosynthesis of an antiviral compound using a stabilized phosphopentomutase by multipoint covalent immobilization.

Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.

Stabilization by multipoint covalent attachment of a biocatalyst with polygalacturonase activity used for juice clarification.

Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity.