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Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

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Abstract

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The Km and Vmax values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni2+, Al3+, and Fe2+ and was increased in the presence of Ca2+ and Mg2+ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.

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Abbreviations

ANOVA:

Analysis of variance

BSA:

Bovine serum albumin

DEPC:

Diethyl pyrocarbonate

DNS:

Dinitrosalicylic acid

DNTB:

5,5′-Dithiobis(2-nitrobenzoic acid)

DTP:

Dithiodipyridine

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

LSD:

Least significant difference

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Acknowledgments

We gratefully appreciate the financial support of this work by the Ministry of Science, Technology and Innovation of Malaysia through Science Fund (02-01-04-SF1800). We would like to thank the staff of the Enzyme Laboratory in Food Science and Technology Faculty of University Putra Malaysia for their help, support, and the use of all facilities which were needed in conducting this study.

Conflict of interest

Mehrnoush Amid and Yazid Manap have received research grant from Ministry of Science, Technology and Innovation of Malaysia (Grant No.02-01-04-SF1800). Khanani Zohdi declares that she has no conflict of interest.

Compliance with Ethics Requirements

This article does not contain any studies with human or animal subjects.

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Authors and Affiliations

  1. Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia (UPM), 43400, UPM Serdang, Selangor, Malaysia

    Mehrnoush Amid, Yazid Manap & Khanani Zohdi

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  1. Mehrnoush Amid
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  2. Yazid Manap
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  3. Khanani Zohdi
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Correspondence to Mehrnoush Amid.

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Amid, M., Manap, Y. & Zohdi, K. Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus . Eur Food Res Technol 239, 21–29 (2014). https://doi.org/10.1007/s00217-014-2188-x

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  • DOI: https://doi.org/10.1007/s00217-014-2188-x

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