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. 2023 Apr 28;33(4):533-542.
doi: 10.4014/jmb.2211.11003. Epub 2022 Dec 30.

Heterologous Expression and Characterization of a Novel Exo-Polygalacturonase from Aspergillus fumigatus Af293 and Its Application in Juice Extraction

Chengwei Yang  1   2 , Ting Zhang  1 , Jing Zhu  1 , Yunyi Wei  1 , Furong Zhu  1 , Zhong Cheng  1
Affiliations

Heterologous Expression and Characterization of a Novel Exo-Polygalacturonase from Aspergillus fumigatus Af293 and Its Application in Juice Extraction

Chengwei Yang et al. J Microbiol Biotechnol. .

Abstract

Exo-polygalacturonase (exo-PG) hydrolyzes pectin acids and liberates mono-galacturonate, which plays an important role in juice extraction, and has rarely been reported. Exo-PG (AfumExoPG28A) from Aspergillus fumigatus belongs to the glycoside hydrolase 28 family. In this study, its gene was cloned and the protein was expressed and secreted in Pichia pastoris with a maximal activity of 4.44 U/ml. The optimal temperature and pH of AfumExoPG28A were 55°C and 4.0, respectively. The enzyme exhibited activity over almost the entire acidic pH range (>20.0% activity at pH 2.5-6.5) and remained stable at pH 2.5-10.0 for 24 h. The Km and Vmax values of AfumExoPG28A were calculated by the substrate of polygalacturonic acid as 25.4 mg/ml and 23.6 U/mg, respectively. Addition of AfumExoPG28A (0.8 U/mg) increased the light transmittance and juice yield of plantain pulp by 11.7% and 9%, respectively. Combining AfumExoPG28A (0.8 U/mg) with an endo-PG (0.8 U/mg) from our laboratory, the enzymes increased the light transmittance and juice yield of plantain pulp by 45.7% and 10%, respectively. Thus, the enzyme's potential value in juice production was revealed by the remarkable acidic properties and catalytic activity in fruit pulp.

Keywords: Aspergillus fumigatus; Exo-polygalacturonase; acid stability; juice extraction.

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Conflict of interest statement

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Figures

Fig. 1
Fig. 1. Multiple sequence alignment of AfumExoPG28A with other functionally characterized GH28 proteins.
Amino acid sequence alignment of AfumExoPG28A, TePG28a (from T. leycettanus., GenBank Accession No. KY474617), CluPG1 (from C. lupini., GenBank Accession No. 2IQ7), endo-PGI (from Penicillium sp., GenBank Accession No. AEL22832), PG7fn (from T. arenaria., GenBank Accession No. AIZ95162), PgaII (from A. niger., GenBank Accession No. CAA41694), T1PGA (from E. leycettana., GenBank Accession No. QKK82827), and ThPG1 (from T. lixii., GenBank Accession No. CAM07141.1), using DNAMAN software. Amino acid residues showing 100%, >75%, and >50% identity are shaded in light black, light red, and light green, respectively. The signal peptide of AfumExoPG28A is underlined. Asp211, Asp231, Asp232, and His255 of AfumExoPG28A, which were expected to be the catalytic residues, are marked by solid circles. The Arg290 and Lys292 of AfumExoPG28A, which were expected to be involved in substrate binding, are marked by solid regular triangles.
Fig. 2
Fig. 2. Time course of exo-polygalacturonase production by P. pastoris.
Fig. 3
Fig. 3. SDS-PAGE analysis of AfumExoPG28A.
Lane 1, molecular weight ladder; lane 2, purified recombinant protein; lane 3, purified recombinant protein digested by EndoH.
Fig. 4
Fig. 4. Substrate specificity of AfumExoPG28A.
PGA, polygalacturonate acid; CP, low-esterified citrus pectin; AP, high-esterified apple pectin; CMC, carboxymethyl cellulose.
Fig. 5
Fig. 5. Michaelis-Menten kinetic parameters of AfumExoPG28A.
The kinetic parameters of the purified AfumExoPG28A were determined by measuring the reaction rates on different concentrations of PGA ranging from 0.20% to 1.0% under the optimal conditions.
Fig. 6
Fig. 6. The effects of pH and temperature on the activity of recombinant AfumExoPG28A.
(A) Effect of pH on recombinant enzyme activity. (B) Effect of pH on enzyme stability. (C) Effect of temperature on recombinant enzyme activity. (D) Effect of temperature on enzyme stability.
Fig. 7
Fig. 7. TLC analysis of purified AfumExoPG28A from A. fumigatus Af293.
Lane 1, galacturonic acid; lanes 2 to 8, liberated products of PGA by AfumExoPG28A for 0, 0.25, 0.5, 1, 2, 8, and 12 h.
See this image and copyright information in PMC

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