High-level secretory expression and characterization of the recombinant Kluyveromyces marxianus inulinase

Zhang, Sufang; Yang, Fan; Wang, Qian; Ye, Hua; Zhao, Zongbao K.

doi:10.1016/j.procbio.2011.10.002

Cited by 23 publications

(17 citation statements)

References 34 publications

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“…However, the molecular mass of the expressed KcINU1 was 90 kDa, 30 kDa larger than the predicted molecular mass (60.6 kDa) of the enzyme. This may be due to post-translational modification of the enzyme, such as glycosylation, carried out by P. pastoris X-33 since inulinase is known to be glycosylated [5,6]. The inulinase activity of the culture supernatant reached 45.2±0.6 U mL −1 after 96 h of induction by methanol.…”

Section: Expression and Purification Of Kcinu1mentioning

confidence: 99%

“…In recent years, inulin has attracted much attention as a renewable raw material for the production of high-fructose syrups, fructoligosaccharides, and ethanol [4]. Inulin can be degraded into fructose by acid treatment (pH=1.0-2.0 at 80-100°C) or enzymatic hydrolysis [5,6]. However, fructose tends to form by-products under acidic conditions, such as 5-hydroxymethylfurfural, which has no sweetening property and is an inhibitor of microbial growth.…”

Section: Introductionmentioning

confidence: 99%

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

Cao

Tan

et al. 2015

Appl Biochem Biotechnol

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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.

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Section: Expression and Purification Of Kcinu1mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Cao

Tan

et al. 2015

Appl Biochem Biotechnol

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“…For investigating effects of the aeration rate on the fermentation by Y179 via CBP, 0, 5 or 10 h À1 (space velocity) was controlled in the experiments. To examine the effect of the more inulinase, 10 U cm À3 of inulinase, which was kindly presented by Professor Du from a recombinant K. marxianus [14], was added into the initial medium (Strategy 1, S1). In the experiment of increased biomass concentration, cells grown in 1.0 L YPD medium was collected, after washing the cells with distilled water, were inoculated into the fermentation medium (Strategy 2, S2).…”

Section: Culture Conditionsmentioning

confidence: 99%

Efficient ethanol production from inulin by two-stage aerate strategy

Gao¹,

Yuan²,

Kong³

et al. 2015

Biomass and Bioenergy

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“…This suggests that molecular mass of the exoinulinases from bacteria is almost the same as of the exoinulinases from yeasts. K. marxianus CBS 6556 inulinase (rKmINU) gene expressed in methylotrophic host Pichia pastoris showed a specifi c activity of 2714 U/mg (29), which is 12-fold higher than those of other inulinases described previously. It displayed excellent stability from 30 to 50 °C and pH=3.0-5.0, and its half-life was over 96 h under these conditions.…”

Section: Purifi Cation Of Inulinasementioning

confidence: 63%

“…It displayed excellent stability from 30 to 50 °C and pH=3.0-5.0, and its half-life was over 96 h under these conditions. Moreover, rKmINU saccharifi ed Jerusalem artichoke tuber juice eff ectively (29). A 79.8-kDa endoinulinase gene (enIA) from Arthrobacter sp.…”

Section: Purifi Cation Of Inulinasementioning

confidence: 99%

Purification and Characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant

Naidoo

Kumar

Sharma

et al. 2015

Food Technol. Biotechnol.

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SummaryAn extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purifi ed to homogeneity by gel fi ltration chromatography and showed a specifi c activity of 119 U/mg. The optimum pH and temperature of the purifi ed enzyme were found to be 6.0 and 50 °C, respectively. The enzyme was stable up to 60 °C, retaining 60 % of residual activity for 30 min, but inactivated rapidly above 60 °C. The enzyme was found to be stable at pH=6-9 when it retained 100 % of its residual activity. The Lineweaver-Burk plot showed that the apparent K m and v max values of the inulinase when using inulin as a substrate were 1.15 mg/mL and 0.15 μM/min, respectively, whereas the k cat value was found to be 0.145 min -1 . The calculated catalytic effi ciency of the enzyme was found to be 0.126 (mg·min)/mL. The purifi ed inulinase can be used in the production of high fructose syrups.

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High-level secretory expression and characterization of the recombinant Kluyveromyces marxianus inulinase

Cited by 23 publications

References 34 publications

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

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

Efficient ethanol production from inulin by two-stage aerate strategy

Purification and Characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant

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