NDLI: Enhancement of novel Endo-polygalacturonase expression in Rhodotorula mucilaginosa PY18: insights from mutagenesis and molecular docking

Content Provider	Springer Nature : BioMed Central
Author	Abd El-Aziz, Nagwa M. Moharam, Maysa E. El-Gamal, Nora N. Khalil, Bigad E.
Abstract	Pectinase is a particular type of enzyme that can break down pectin compounds and is extensively utilised in the agricultural field. In this study, twenty yeast isolates were isolated and assayed for pectinase activity. Molecular identification by PCR amplification and sequencing of internal transcribed spacer (ITS) regions of isolate no. 18 had the highest pectinase activity of 46.35 U/mg, was identified as Rhodotorula mucilaginosa PY18, and was submitted under accession no. (OM275426) in NCBI. Rhodotorula mucilaginosa PY18 was further enhanced through sequential mutagenesis, resulting in a mutant designated as Rhodotorula mucilaginosa E54 with a specific activity of 114.2 U/mg. Using Response Surface Methodology (RSM), the best culture conditions for the pectinase-producing yeast mutant Rhodotorula mucilaginosa E54 were pH 5, 72-h incubation, 2.5% xylose, and 2.5% malt extract, with a pectinase-specific activity of 156.55 U/mg. Then, the obtained sequences of the endo-polygalacturonase PGI gene from Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were isolated for the first time, sequenced, and submitted to NCBI accession numbers OQ283005 and OQ283006, respectively. The modelled 3D structure of the endo-PGI enzyme (485 residues) was validated using Ramachandran’s plot, which showed 87.71, 85.56, and 91.57% in the most favourable region for template Rhodotorula mucilaginosa KR, strain Rhodotorula mucilaginosa PY18, and mutant Rhodotorula mucilaginosa E54, respectively. In molecular docking studies, the results of template Rhodotorula mucilaginosa KR endo-PG1 showed an interaction with an affinity score of − 6.0, − 5.9, and − 5.6 kcal/mol for active sites 1, 2, and 3, respectively. Rhodotorula mucilaginosa PY18 endo-PG1 showed an interaction affinity with a score of − 5.8, − 6.0, and − 5.0 kcal/mol for active sites 1, 2, and 3, respectively. Mutant Rhodotorula mucilaginosa E54 endo-PG1 showed an interaction affinity of − 5.6, − 5.5, – 5.5 and − 5.4 kcal/mol for active sites 1, 2, and 3, respectively. The endo-PGI genes of both the yeast strain Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were successfully cloned and expressed in E. coli DH5α, showing significantly higher endo-PG1 activity, which recorded 94.57 and 153.10 U/mg for recombinant Rhodotorula mucilaginosa pGEM-PGI-PY18 and recombinant mutant Rhotorula pGEM-PGI-E54, respectively.
Related Links	https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-023-02253-5.pdf
Ending Page	23
Page Count	23
Starting Page	1
File Format	HTM / HTML
ISSN	14752859
DOI	10.1186/s12934-023-02253-5
Journal	Microbial Cell Factories
Issue Number	1
Volume Number	22
Language	English
Publisher	BioMed Central
Publisher Date	2023-12-08
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Rhodotorula mucilaginosa PY18 Pectinase Mutagenesis Endo-polygalacturonase (endo-PGI) gene Cloning and expression Modelled 3D structure Molecular docking
Content Type	Text
Resource Type	Article
Subject	Applied Microbiology and Biotechnology Bioengineering Biotechnology
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.5/2023

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