Novel thermophilic pectinases for recycling sustainable biomass

Abstract

Large amounts of pectin-rich biomass are produced mainly from juice and sugar industries. Pectinases represent an economical and eco-friendly alternative to depolymerise it in comparison with chemical treatments, for both obtaining bio-based chemicals and improving industrial bioprocess. This thesis aimed to identify novel thermophilic pectinases, carry out their functional characterisation, and explore the synergistic activity between pectin methylesterases (PMEs) and exo-polygalacturonases (exo-PGs) for pectin bioconversion into galacturonic acid (GalA). Also, the work was focused on the co-expression of genes encoding a PME and exo-PG in a single host for a cost-effective pectin bioconversion into GalA. Finally, the synergistic activity between PMEs and pectate lyases (PGLs) for improving pectin depolymerisation, useful in several industrial processes, was also explored. A total of seven genes encoding thermophilic bacterial pectinases were successfully cloned, and the enzymes expressed included two exo-PGs (TMA01 and BLI04), two PMEs (BLI09 and SAM10) and three PGLs (TMA14, TFU19 and TFU20). Mn²⁺ significantly increased the exo-PGs activity (2-fold) and did not affect PMEs action allowing its inclusion in the synergistic reactions. Both exo-PGs and PMEs exhibited high activity and stability between 40 and 90 °C up to 24 h. The synergistic reactions between BLI09 PME paired either with TMA01 or BLI04 exo-PGs using apple and citrus pectin were the most successful for pectin bioconversion, releasing around 2.5 mM GalA (29% yield). GalA release by pectinases is important in industry since it is a key chemical for the synthesis of a number of valuable compounds. In addition, enzymes allow to release this compound in a sustainable biocatalytic process. Four co-expression plasmids containing a PME and exo-PG were constructed in pETDuet-1, in which the gene’s cloning order as well as the presence of a T7 terminator behind the second gene affected the pectinases expression levels. TMA01 and BLI04 exo-PGs were well expressed in all the constructs, but BLI09 PME was better expressed cloned downstream the exo-PGs in MCS-2 and with the presence of its own T7 terminator behind. Thus, the most successful co-expression plasmids were the constructs 3 and 4 (pETDuet-TMA01-BLI09 and 4 pETDuet-BLI04-BLI09, respectively) which allowed the release of around 3 mM GalA (35% yield) from apple and citrus pectin. GalA release was limited by product inhibition since this compound had an inhibitory effect on both exo-PGs from around 3 mM. Finally, the three PGLs were characterised showing Ca²⁺ dependant activity, while Mn2+ significantly improved the activity of TFU20 (2.5-fold). All the PGLs exhibited optimum activity and stability between 50 and 90 °C and Ca²⁺ improved their thermal stability. The three PGLs were able to depolymerise pectin, but the main peak of 650 kDa observed in both non-esterified and esterified substrates was depolymerised only in the non-esterified. These results evidenced the need of a synergistic action of PGLs with PMEs for improving esterified pectin depolymerisation. Thus, the smallest oligogalacturonates (oligoGalA) because of this main peak depolymerisation in apple, citrus and sugar beet pectin were obtained from synergistic reactions between SAM10 PME paired either with TMA14 (4 – 10 kDa) or TFU20 (8 – 25 kDa). Overall, this work provided optimum conditions of activity of novel thermophilic pectinases, which are fundamental to set up compatible operational conditions especially in synergistic reactions. Synergistic activity between pectinases allowed the release of GalA and co-expression systems made the pectin bioconversion process more cost-effective. Synergistic activity also improved esterified pectin depolymerisation, useful and applicable to several industries. These findings provided further insights for recycling sustainable biomass within a context of biorefineries and circular economy.

Se clonaron con éxito un total de siete genes que codifican pectinasas bacterianas termófilas y las enzimas expresadas incluyeron dos exo-PG (TMA01 y BLI04), dos PME (BLI09 y SAM10) y tres PGL (TMA14, TFU19 y TFU20). Mn⁺² aumentó significativamente la actividad de las exo-PG (2 veces) y no afectó la acción de las PME, lo que permitió su inclusión en las reacciones sinérgicas. Tanto las exo-PG como las PME exhibieron alta actividad y estabilidad entre 40 y 90 °C hasta 24 h. Las reacciones sinérgicas entre BLI09 PME con exo-PG TMA01 o BLI04 usando pectina de manzana y cítrica fueron las más exitosas para la bioconversión de pectina, liberando alrededor de 2,5 mM de GalA (rendimiento del 29%). La liberación de GalA por las pectinasas es importante en la industria, ya que es una sustancia química clave para la síntesis de varios compuestos valiosos. Además, las enzimas permiten liberar este compuesto en un proceso biocatalítico sostenible. En pETDuet-1 se construyeron cuatro plásmidos de coexpresión que contenían una PME y exo-PG, en los que el orden de clonación del gen, así como la presencia de un terminador T7 detrás del segundo gen, afectaron los niveles de expresión de las pectinasas. Las exo-PG de TMA01 y BLI04 se expresaron bien en todas las construcciones, pero la PME de BLI09 se expresó mejor clonada después de las exo-PG en MCS-2 y con la presencia de su propio terminador T7. Por lo tanto, los plásmidos de coexpresión más exitosos fueron las construcciones 3 y 4 (pETDuet-TMA01-BLI09 y pETDuet-BLI04-BLI09, respectivamente) que permitieron la liberación de alrededor de 3 mM de GalA (35% de rendimiento) de pectina de manzana y cítrica. La liberación de GalA estuvo limitada por la inhibición del producto, ya que este compuesto tuvo un efecto inhibidor sobre ambas exo-PG desde aproximadamente 3 mM. Finalmente, las tres PGL mostraron actividad dependiente de Ca⁺² , mientras que Mn⁺² mejoró significativamente la actividad de TFU20 (2,5 veces). Todas las PGL exhibieron actividad y estabilidad óptimas entre 50 y 90 °C y Ca⁺² mejoró su estabilidad térmica. Las tres PGL pudieron despolimerizar la pectina, pero el pico principal de 650 kDa observado tanto en los sustratos esterificados como no esterificados se despolimerizó solo en los no esterificados. Estos resultados evidenciaron la necesidad de una acción sinérgica de las PGL con las PME para mejorar la despolimerización de la pectina esterificada. Así, los oligogalacturonatos más pequeños producto de la despolimerización de este pico principal en pectina de manzana, cítrica y remolacha azucarera se obtuvieron a partir de reacciones sinérgicas entre SAM10 PME con TMA14 (4 – 10 kDa) o TFU20 (8 – 25 kDa).