Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris
Bacterial lipase poses potential challenges when expressed in eukaryotic protein expression systems such as Pichia pastoris. This research aims to increase extracellular T1.2RQ lipase secretion (free lipase) with multiple gene copy number strategies in Pichia pastoris and it was first performed on...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Bogor Agricultural University
2024-12-01
|
| Series: | Hayati Journal of Biosciences |
| Online Access: | https://journal.ipb.ac.id/index.php/hayati/article/view/57548 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1841554891893374976 |
|---|---|
| author | Esti Puspitasari Yepy Hardi Rustam Andreas Adhi Satya Antonius Suwanto Aris Tri Wahyudi Rika Indri Astuti |
| author_facet | Esti Puspitasari Yepy Hardi Rustam Andreas Adhi Satya Antonius Suwanto Aris Tri Wahyudi Rika Indri Astuti |
| author_sort | Esti Puspitasari |
| collection | DOAJ |
| description |
Bacterial lipase poses potential challenges when expressed in eukaryotic protein expression systems such as Pichia pastoris. This research aims to increase extracellular T1.2RQ lipase secretion (free lipase) with multiple gene copy number strategies in Pichia pastoris and it was first performed on lipase from Geobacillus stearothermophilus T1.2. In this study, the T1.2RQ lipase gene from Geobacillus stearothermophilus T1.2 was expressed in Pichia pastoris GS115 through a strategy involving multiple copies of lipase, resulting in increased lipase activity. Three copies of the lipase gene in pPIC9K_T1.2RQ(3x) recombinant plasmid were integrated into the genome of Pichia pastoris GS115, and quantitative analysis using qPCR technique confirmed that the GS115 transformant strain contained six copies of T1.2RQ gene, indicating two integration events. Lipase activity measurement showed that the GS115/T1.2RQ(6x) strain exhibited a 111% increase compared to that containing a copy of the T1.2RQ gene. SDS-PAGE and Zymogram results showed a protein band with a size of 43kDa. Qualitative analysis in LA+TBN media of all strains containing the T1.2RQ gene showed clear zones. Lipase production in flask fermentation took at least 120 hours to produce the best lipase activity. Thus, strategies with multiple copy numbers of gene lipase have significantly increased the expression of the bacterial lipase gene in Pichia pastoris GS115.
|
| format | Article |
| id | doaj-art-bb3177b56bb349e387c8022c5d21d8d7 |
| institution | Kabale University |
| issn | 1978-3019 2086-4094 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Bogor Agricultural University |
| record_format | Article |
| series | Hayati Journal of Biosciences |
| spelling | doaj-art-bb3177b56bb349e387c8022c5d21d8d72025-01-08T09:00:49ZengBogor Agricultural UniversityHayati Journal of Biosciences1978-30192086-40942024-12-0132210.4308/hjb.32.2.310-319Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris Esti Puspitasari0Yepy Hardi Rustam1Andreas Adhi Satya2Antonius Suwanto3Aris Tri Wahyudi4Rika Indri Astuti5Department of Research and Development Biotechnology, PT Wilmar Benih Indonesia, Cikarang 17530, IndonesiaDepartment of Research and Development Biotechnology, PT Wilmar Benih Indonesia, Cikarang 17530, IndonesiaDepartment of Research and Development Biotechnology, PT Wilmar Benih Indonesia, Cikarang 17530, IndonesiaDepartment of Biology, Faculty of Mathematics and Natural Science, IPB University, Bogor 16680, IndonesiaDepartment of Biology, Faculty of Mathematics and Natural Science, IPB University, Bogor 16680, IndonesiaDepartment of Biology, Faculty of Mathematics and Natural Science, IPB University, Bogor 16680, Indonesia Bacterial lipase poses potential challenges when expressed in eukaryotic protein expression systems such as Pichia pastoris. This research aims to increase extracellular T1.2RQ lipase secretion (free lipase) with multiple gene copy number strategies in Pichia pastoris and it was first performed on lipase from Geobacillus stearothermophilus T1.2. In this study, the T1.2RQ lipase gene from Geobacillus stearothermophilus T1.2 was expressed in Pichia pastoris GS115 through a strategy involving multiple copies of lipase, resulting in increased lipase activity. Three copies of the lipase gene in pPIC9K_T1.2RQ(3x) recombinant plasmid were integrated into the genome of Pichia pastoris GS115, and quantitative analysis using qPCR technique confirmed that the GS115 transformant strain contained six copies of T1.2RQ gene, indicating two integration events. Lipase activity measurement showed that the GS115/T1.2RQ(6x) strain exhibited a 111% increase compared to that containing a copy of the T1.2RQ gene. SDS-PAGE and Zymogram results showed a protein band with a size of 43kDa. Qualitative analysis in LA+TBN media of all strains containing the T1.2RQ gene showed clear zones. Lipase production in flask fermentation took at least 120 hours to produce the best lipase activity. Thus, strategies with multiple copy numbers of gene lipase have significantly increased the expression of the bacterial lipase gene in Pichia pastoris GS115. https://journal.ipb.ac.id/index.php/hayati/article/view/57548 |
| spellingShingle | Esti Puspitasari Yepy Hardi Rustam Andreas Adhi Satya Antonius Suwanto Aris Tri Wahyudi Rika Indri Astuti Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris Hayati Journal of Biosciences |
| title | Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris |
| title_full | Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris |
| title_fullStr | Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris |
| title_full_unstemmed | Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris |
| title_short | Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris |
| title_sort | effect of multiple gene copy number of bacterial lipase to increase lipase production in pichia pastoris |
| url | https://journal.ipb.ac.id/index.php/hayati/article/view/57548 |
| work_keys_str_mv | AT estipuspitasari effectofmultiplegenecopynumberofbacteriallipasetoincreaselipaseproductioninpichiapastoris AT yepyhardirustam effectofmultiplegenecopynumberofbacteriallipasetoincreaselipaseproductioninpichiapastoris AT andreasadhisatya effectofmultiplegenecopynumberofbacteriallipasetoincreaselipaseproductioninpichiapastoris AT antoniussuwanto effectofmultiplegenecopynumberofbacteriallipasetoincreaselipaseproductioninpichiapastoris AT aristriwahyudi effectofmultiplegenecopynumberofbacteriallipasetoincreaselipaseproductioninpichiapastoris AT rikaindriastuti effectofmultiplegenecopynumberofbacteriallipasetoincreaselipaseproductioninpichiapastoris |