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Recombinant Thermostable Thermomonospora fusca TF Endo-xylanase A and Its Immobilization on Modified Mesoporous SiO2 Microspheres for Manufacturing Xylooligosaccharides

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Abstract

tfxa (GenBank: U01242), which encoded the mature peptide of Thermomonospora fusca TF xylanase A (TfxA), was high-level expressed in Escherichia coli BL21 (DE3). The recombinant xylanase, reETfxA, was both secreted into culture medium and in the cytoplasm. reETfxA showed high xylanase activity and was purified to homogeneity by Ni-affinity resin (182.8 and 681.4 U mg−1 for crude and purified enzyme, respectively). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blot analysis revealed that the molecular mass of reETfxA was approximately 42.7 kDa. The reETfxA was immobilized on a novel mesoporous SiO2 microsphere (MSM)-coated chitosan via covalent bonds, which formed from Schiff base reaction between supports and enzyme. The binding capacity of the prepared MSM-coated chitosan particles to reETfxA was approximately 272.6 mg g−1-particles. The optimum temperature values of the free and immobilized reETfxA were 65 and 70 °C, respectively. Moreover, the optimum pH values of the free and immobilized reETfxA were pH 6.0 and 5.0, respectively. The reETfxA showed relatively high thermostability. When treated at 70 °C and pH 6.0 for 15 min, the residual activities of free and immobilized reETfxA were 84.2 and 100.2%, respectively. The results obtained from the HPLC analysis showed that immobilized reETfxA released xylooligosaccharides from oat spelt, beechwood and birchwood xylans, with xylotetraose, xylotriose, and xylopentaose as the major products, respectively. Additionally, the immobilized reETfxA could directly hydrolyze the wheat bran insoluble xylan.

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Abbreviations

TfxA:

Thermomonospora fusca TF xylanase A

tfxa :

The gene the encoding mature peptide of TfxA

X1:

Xylose

X2:

Xylobiose

X3:

Xylotriose

X4:

Xylotetraose

X5:

Xylopentaose

HPLC:

High–performance liquid chromatography

XOs:

Xylooligosaccharides

\({{K}_{m}}\) :

Michaelis–Menten constants

\({{V}_{\max }}\) :

Maximal activity

MSM:

Mesoporous SiO2 microspheres

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Acknowledgements

This work was supported by the Zhejiang Provincial Natural Science Foundation of China (No. Y15C200013), the National Natural Science Foundation of China (Nos. 31672462, 31271632), and the grant of Science and Technology Department of Zhejiang Province (No. 2016C32086). We thank Dr. Shang-Wei Chen for his kind assistance in the HPLC analysis.

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Authors and Affiliations

  1. National & Local United Engineering Lab of Quality Controlling Technology and Instrumentation for Marine Food, China JiLiang University, Hangzhou, 310018, China

    Ming-qi Liu, Wen-kang Huo & Xin Xu

  2. College of Life Science, Zhejiang University, Hangzhou, 310058, China

    Xiao-yan Weng

  3. College of Biological and Environmental Science, Zhejiang Wanli University, Ningbo, 315100, China

    Qian Wang

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  1. Ming-qi Liu
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  2. Xiao-yan Weng
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  3. Qian Wang
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  4. Wen-kang Huo
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  5. Xin Xu
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Correspondence to Ming-qi Liu.

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Liu, Mq., Weng, Xy., Wang, Q. et al. Recombinant Thermostable Thermomonospora fusca TF Endo-xylanase A and Its Immobilization on Modified Mesoporous SiO2 Microspheres for Manufacturing Xylooligosaccharides. Catal Lett 147, 765–772 (2017). https://doi.org/10.1007/s10562-017-1979-7

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  • DOI: https://doi.org/10.1007/s10562-017-1979-7

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