Primer Design for Amplification of Lignin Peroxidase H8 (lipH8) from Phanerochaete chrysosporium using Ugene Software as an Effort to Accelerate Lignocellulose-based Bioenergy Production Technology

⁴ Biotechnology Program, Department of Applied Sciences, Faculty of Mathematics and Science, State University of Malang, Malang, Indonesia
² Faculty of Vocational , Universitas Airlangga, Surabaya, Indonesia
³ Departement of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Malaysia
¹ Chemistry Program, Faculty of Mathematic and Science, State University of Malang, Malang, Indonesia

^* Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.

Abstract

Ligninase enzymes play a crucial role in the delignification stage of lignocellulose-based bioenergy production, especially in bioethanol production, providing an environmentally friendly and cost-efficient process with minimal byproducts. However, the practical application of this strategy is limited by the low expression level of wild-type ligninase from Phanerochaete chrysosporium. Heterologous protein expression provides a promising solution to overcome this limitation. Primer design is a key step in recombinant DNA assembly for successful heterologous expression. Using bioinformatic tool, in silico primer design enables pre-laboratory optimization to enhance experimental success rate. This study aimed to design primers for the lipH8 gene from Phanerochaete chrysosporium through sequence homology analysis using the Ugene software. Lignin peroxidase isozyme H8 (lipH8) showed the highest catalytic activity in lignin degradation among the ligninase family. Primer selection was based on amplification size, self-dimer formation, and open reading frame consideration. The optimal primer pair identified was a 20-base pair forward primer (‘5-GCATGGTGGGGTGAAATACG-‘3) and reverse primer (‘5-TGTGAGACGAGTCGGTGATG-‘3), predicted to amplify a 1630 bp fragment of lipH8. This primer pair showed no self-dimer formation and exhibited superior open reading frame coverage compared to other candidates, making it suitable for further experimental validation in heterologous expression system.