A Comparative Analysis of the Properties of Recombinant Endoinulinase, Exoinulinase, Sucrase, and Alpha-Galactosidase C

Abstract

As a result of cloning of the inuA, inu1, aglC, and fopA genes encoding endoinulinase (endoINU), exoinulinase (exoINU), α-galactosidase C (AGLС) and sucrase (SUC), respectively, into the recipient strain Penicillium verruculosum B1-537 (ΔniaD), recombinant producer strains were obtained that are capable of producing target recombinant enzymes with a high yield (32‒50% of the total extracellular protein). Enzyme preparations of endoINU, exoINU, AGLC, and SUC were obtained and characterized. Using chromatographic methods, endoINU, exoINU, SUC, and AGLC with a molecular weights of 62, 56, 67, and 76 kDa, respectively, were isolated in a homogeneous form (according to polyacrylamide gel electrophoresis). The homogeneous endoINU had a high specific activity against Jerusalem artichoke inulin (56 U/mg). ExoINU was active towards inulin (17 U/mg), sucrose (850 U/mg), raffinose (41 U/mg), and stachyose (15 U/mg). SUC decomposed sucrose (10.5 U/mg), raffinose, and stachyose (3.8 and 1.4 U/mg, respectively). AGLC had raffinase and stachyase activities (31 U/mg and 30 U/mg, respectively), exhibited no activity towards sucrose, but had a high level of activity towards the synthetic substrate, p-nitrophenyl-α-D-galactoside (311 U/mg). The kinetic parameters (k_cat and K_m) of the hydrolysis of the corresponding substrates by homogeneous enzymes were determined. The temperature optimum was 50‒55°C for endoINU, 55‒65°C for exoINU, 65°C for AGLC, and 35°C for SUC. EndoINU, exoINU, AGLC and SUC exhibited its maximum activity at pH 6.5, 4.5, 4.5‒5.0, and 5.5‒6.0, respectively. The thermal stability of the enzymes was studied at different temperatures. EndoINU exhaustively hydrolyzed inulin with the formation of fructooligosaccharides with a degree of polymerization of 3‒8. ExoINU quantitatively converted inulin into glucose-fructose syrup (GFS) with a Glu : Fru ratio of 1 : 3, and sucrose into GFS with a Glu : Fru ratio of about 1 : 0.63 (SUC provided the same results in the sucrose hydrolysis). Soy galactooligosaccharides (raffinose and stachyose) were converted to sucrose and monosaccharides (glucose, galactose, and fructose) under the action of AGLC. The combined action of SUC, and AGLC resulted in a complete conversion of raffinose, stachyose and sucrose to monosaccharides. The same results were achieved using ExoINU. This enzyme can be considered promising for biotechnological applications due to its broad substrate specificity, which allows it be used both for the production of GFS from inulin and sucrose, and for the destruction of soybean galactooligosaccharides.