Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus

Abstract

Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 μM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase.

The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857

“…In previous study, we have investigated the conserved residues (D30 and E215 in previous paper, in this paper numbered D53 and E238, respectively, including the 23 amino acids of the signal peptide) which involved in catalytic activity through sequences alignments (Ma et al, 2016). However, it should be noted that there was an aromatic region observed around the enzyme-catalyzed site in the GH32 family, which involves the sequences WMNDPN, WG, and W/FSGSAT.…”

“…Plasmid pPICZαA-rKcINU1 containing the kcINU1 gene (GenBank accession number AF178979) without the N-terminal signal peptide coding sequence was preserved in this laboratory (Ma et al, 2016). Phusion Hot Start II DNA polymerase was purchased from Thermo Fisher (China).…”

“…The procedure of zymogram analysis was conducted as previously described (Ma et al, 2016). In this study, sucrose and inulin were used as the substrates.…”

“…The incubation was lasted for 15 min at 37 • C and protected from light, with which visual inspection was performed. The relative enzyme activities were measured spectrophotometrically (540 nm) by a DNS method as referred previously (Ma et al, 2016). As for the kinetic properties for native enzyme and variants, the K m and V max were determined by DNS method using inulin (2.9-52.1 mM) and sucrose (0.2-2.6 mM) as the substrates.…”

“…After affinity chromatography Ni affinity column, the obtained variants were analyzed using SDS-PAGE, as shown in Figure 3A. The targeted proteins have clear bands around 90 kDa due to glycosylations (their predicted molecular mass were around 60.6 kDa) (Ma et al, 2016).…”