Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162

Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162 Publisher Pubmed

Jafari F¹ ; Kianighaleh F² ; Eftekhari S³ ; Razzaghshoar Razlighi M⁴ ; Nazari N⁵ ; Hajirajabi M⁶ ; Masoomi Sarvestani F⁷ ; Sharafieh G^{8, 9}

Show Affiliations

1. Molecular Biotechnology Laboratory, Department of Biology, Faculty of Science, Shiraz University, Shiraz, Iran
2. Department of Chemical Engineering, Shahreza Branch, Islamic Azad University, Shahreza, Iran
3. Medical Laboratory Sciences Department, Islamic Azad University, Tehran Medical Branch, Tehran, Iran
4. Department of Biology, East Tehran Branch, Islamic Azad University, Tehran, Iran
5. Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran
6. Department of Microbiology, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran
7. Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
8. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
9. Department of Clinical Biochemistry, Islamic Azad University, Tehran, Iran

Source: Preparative Biochemistry and Biotechnology Published:2022

Abstract

The main purpose of the present study is to introduce the biochemical characteristics of the industrial valuable thermostable pullulan degrading enzyme from Desulfurococcus mucosus DSM2162. Recombinant protein was purified by a combination of thermal treatment and affinity chromatography, with a yield of 15.94% and 7.69-fold purity. Purified enzyme showed the molecular mass of 55,787 Da with optimum activity at 70 °C and a broad range of pH (5.0–9.0) with kcat of 2150 min−1 and Km of 6.55 mg.mL−1, when using starch as substrate. The enzyme activity assay on various polysaccharide substrates revealed the substrate preference of pullulan > amylopectin > β cyclodextrin > starch > glycogen; therefore, it classified as a neopullulanase. The neopullulanase structural analysis by spectrofluorometer, FT-IR, and circular dichroism spectroscopy indicated the corporation of α-helix (47.3%) and β-sheet (31.6%) in its secondary structure. The melting temperature and specific heat capacity calculations using differential scanning calorimetry confirmed its extreme thermal stability. Further, salt-elevated concentrations resulted in oligomeric state dominancy without any significant influence on the starch-degrading ability. The newly cloned archaeal neopullulanase was with broad activity on polysaccharide substrates, with thermal and salt stability. Thus, the Desulfurococcus mucosus DSM2162 neopullulanase can be introduced as a good candidate to be used in carbohydrate industry. © 2022 Taylor & Francis Group, LLC.

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Style	Citing Format
MLA	Jafari F, et al.. "Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162." Preparative Biochemistry and Biotechnology, vol. 52, no. 10, 2022, pp. 1190-1201.
APA	Jafari F, Kianighaleh F, Eftekhari S, Razzaghshoar Razlighi M, Nazari N, Hajirajabi M, Masoomi Sarvestani F, Sharafieh G (2022). Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162. Preparative Biochemistry and Biotechnology, 52(10), 1190-1201.
Chicago	Jafari F, Kianighaleh F, Eftekhari S, Razzaghshoar Razlighi M, Nazari N, Hajirajabi M, Masoomi Sarvestani F, Sharafieh G. "Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162." Preparative Biochemistry and Biotechnology 52, no. 10 (2022): 1190-1201.
Harvard	Jafari F et al. (2022) 'Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162', Preparative Biochemistry and Biotechnology, 52(10), pp. 1190-1201.
Vancouver	Jafari F, Kianighaleh F, Eftekhari S, Razzaghshoar Razlighi M, Nazari N, Hajirajabi M, et al.. Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162. Preparative Biochemistry and Biotechnology. 2022;52(10):1190-1201.
BibTex	@article{ author = {Jafari F and Kianighaleh F and Eftekhari S and Razzaghshoar Razlighi M and Nazari N and Hajirajabi M and Masoomi Sarvestani F and Sharafieh G}, title = {Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162}, journal = {Preparative Biochemistry and Biotechnology}, volume = {52}, number = {10}, pages = {1190-1201}, year = {2022} }
RIS	TY - JOUR AU - Jafari F AU - Kianighaleh F AU - Eftekhari S AU - Razzaghshoar Razlighi M AU - Nazari N AU - Hajirajabi M AU - Masoomi Sarvestani F AU - Sharafieh G TI - Cloning, Overexpression, and Structural Characterization of a Novel Archaeal Thermostable Neopullulanase From Desulfurococcus Mucosus Dsm 2162 JO - Preparative Biochemistry and Biotechnology VL - 52 IS - 10 SP - 1190 EP - 1201 PY - 2022 ER -

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