Extraction and characterization of β-fructofuranosidases produced by Kluyveromyces marxianus CCMB 322

Invertase (β-fructofuranosidase, EC 3.2.1.26) catalyzes sucrose hydrolysis into glucose and fructose and it is one of the simplest carbohydrases. These enzymes occur widely in nature and their presence has been reported in microorganisms and plants. Since yeasts are the main industrial source, most researches concerning this enzyme have focused on invertase extracted from such source. This study extracted and characterized inverted intracellular (Inv-I) and extracellular (Inv-E) of Kluyveromyces marxianus CCMB 322 isolated in the baiano semi-arid region. Kluyveromyces marxianus CCMB 322 produces intracellular and extracellular invertase with different characteristics. The optimum activity was achieved at approximately pH 3.9 and 45ºC, in Inv-I and Inv-E. The invertases produced by K. marxianus CCMB 322 showed thermal stability similar to that found in other studies. The Km and Vmax values of the Inv-I enzyme were 61.12mM and 5.56 µmol/mL.min-1, but the Km and Vmax values of the Inv-E enzyme were 76.5mM and 0.364 µmol/mL.min-1. Inverted from K. marxianus is a higher affinity for sucrose compared to enzymes from other sources.

Physiological basis for enhanced sucrose accumulation in an engineered sugarcane cell line

Transgenic sugarcane (Saccharum officinarum L. interspecific hybrids) line N3.2 engineered to express a vacuole-targeted sucrose isomerase was found to accumulate sucrose to twice the level of the background genotype Q117 in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose levels declined over successive subcultures, but the enhanced sucrose accumulation was stable. Detailed physiological characterisation revealed multiple processes altered in line N3.2 in a direction consistent with enhanced sucrose accumulation. Striking differences from the Q117 control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose-cleavage enzymes (particularly sucrose synthase breakage activity), and enhanced levels of symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate (G6P). Sucrose-non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates sucrose synthase breakage activity while downregulating SPS in plants) was significantly lower in line N3.2 during the period of fastest sucrose accumulation. For the first time, T6P is also shown to be a negative regulator of SnRK1 activity from sugarcane sink cells, hinting at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane.