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Enhanced Lipase Production in Pichia pastoris via Multiple Copies of Bacterial Lipase Genes and Co-expression of the HAC1 Gene Puspitasari, Esti; Rustam, Yepy Hardi; Satya, Andreas Adhi; Suwanto, Antonius; Wahyudi, Aris Tri; Astuti, Rika Indri
HAYATI Journal of Biosciences Vol. 33 No. 2 (2026): March 2026
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.33.2.288-296

Abstract

The hac1 gene, a key regulator of the untranslated protein response (UPR), was co-expressed in Pichia pastoris GS115 to enhance the production of a lipase from Geobacillus stearothermophilus. Multicopy lipase constructs (1X and 4X) were transformed with pPICZAwbe_hac1, generating GS115/T1.2RQ(1X)_hac1 and GS115/T1.2RQ(4X)_hac1 strains. The GS115/T1.2RQ(1X)_hac1 strain showed an 186% lipase activity after 120 hours versus the control (100%), while the GS115/T1.2RQ(4X)_hac1 strain showed a faster initial increase (38% at 48 hours) and 28% at 120 hours, which was beneficial for efficient enzyme production. Overexpression of the hac1 gene enhances lipase production because it activates UPR genes when the endoplasmic reticulum is stressed due to a large number of recombinant proteins and forms proteins that are not appropriately folded. SDS-PAGE and tributyrin plate assays confirmed extracellular lipase expression (~43 kDa). These results demonstrate that hac1 co-expression significantly (p = 0.01)  enhances lipase production in Pichia pastoris, especially in lower-copy constructs. This is the first report of co-expressing hac1 with Geobacillus stearothermophilus lipase genes in yeast. The findings are expected to contribute to developing more efficient microbial cell factories for producing industrial enzymes.
Identification of nifD and nifH Genes of Methanotrophic Bacteria from Rice Field Bintarti, Ari Fina; Rusmana, Iman; Wahyudi, Aris Tri
Annales Bogorienses Vol. 18 No. 2 (2014): Annales Bogorienses
Publisher : BRIN

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Abstract

Metanotrophic bacteria have ability to oxidize methane and fix atmospheric nitrogen, hence the bacteria has an important role as a nitrogen source provider on wetland area like rice fields. Nitrogen fixation process is catalyzed by the nitrogenase enzyme complex, encoded by nifD and nifH genes. However, characteristic of these genes from indigenous-methanotrophic bacteria still poorly understood. Hence, nifD and nifH genes of methanotrophic bacteria isolated from rice fields in Indonesia (BGM3, BGM9, SS1, SS3, SS10, ST18, SP3, and INP4) were identified and characterized. Detection of nifH and nifD genes was conducted by polymerase chain reaction (PCR) amplification. nifH and nifD gene sequences were analyzed using BLAST-X and phylogenetic trees were constructed using Neighbour Joining method. Based on nifH sequences analysis, SS1 closely related to Beijerinckia mobilis and SS3, SS10, ST 18 closely related to Beijerinckia indica subsp. indica ATCC 9039, while, BGM3, INP4, and BGM9 related to nifH of uncultured nitrogen-fixing bacterium. In other hand, sequence analysis of nifD gene showed that SS1, SS3, SS10, ST 18 closely related to B. indica subsp. indicaATCC 9039 and BGM3, BGM9, INP4 closely related to Xanthobacter autotrophicus Py2. Identification by 16S rRNA gene indicated that SS1, SS3, SS10, and ST18 had closeness to Beijerinckia sp. P310-1, while INP4 closely related to Xanthobacter sp. M5C24.
Phenotypic and Genotypic Characterization of Bacteria from Diabetic Ulcers: Insights into Resistance and Biofilm Formation Ernawati, Andi; Rusmana, Iman; Wahyudi, Aris Tri; Murtini, Sri; Budiarti, Sri
Journal of Multidisciplinary Applied Natural Science Articles in Press
Publisher : Pandawa Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.47352/jmans.2774-3047.348

Abstract

Diabetic ulcers are prone to colonization by multidrug-resistant bacteria, complicating treatment and recovery. Therefore, understanding the microbial profile and resistance mechanisms is essential for effective clinical management. This study isolated and characterized bacteria from diabetic ulcer patients in West Java, Indonesia. Identification was performed using the selective differential medium test and 16S rRNA sequencing. Antibiotic susceptibility was assessed via the disc diffusion method, and biofilm formation was evaluated using crystal violet staining. PCR amplification targeted blaTEM and sul1 resistance genes. A total of 15 bacterial isolates were identified, predominantly Staphylococcus aureus and Pseudomonas aeruginosa. Six isolates exhibited a multiple antibiotic resistance (MAR) index >0.4 and demonstrated biofilm-forming capacity. The PCR result confirmed the presence of blaTEM and sul1 genes in three isolates. The findings highlight the prevalence of multidrug-resistant, biofilm-forming bacteria in diabetic ulcers, underscoring the need for targeted antimicrobial strategies and resistance monitoring.
Modification of a Gene Cassette to Express Proglargine in Pichia pastoris: Elimination of Glu-Ala Repeats, Spacer and C-peptide Sequences Nasution, Uli Julia; Astuti, Rika Indri; Wahyudi, Aris Tri; Hardianto, Dudi; Martius, Efrida
Journal of Multidisciplinary Applied Natural Science Articles in Press
Publisher : Pandawa Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.47352/jmans.2774-3047.368

Abstract

The growing global prevalence of diabetes mellitus has sharply increased the demand for insulin and its analogues. Pichia pastoris is a well-established system for recombinant pro-insulin and its analogues production. However, conventional gene constructs often include additional sequences, such as Glu-Ala repeats, spacer peptides, and c-peptides that complicate downstream processing and reduce efficiency. This study aimed to construct and express a proglargine (PG) gene cassette lacking the Glu-Ala repeats, spacer, and c-peptide in P. pastoris GS115 to obtain a uniform PG protein. The recombinant vector propagated in Escherichia coli TOP10F’, then expressed in P. pastorisGS115. Selected transformants were cultivated in YPG medium, then induced with 1% and 2% methanol daily in BMMY. The optimum methanol concentration further evaluated in ½ BSM induction medium. The result demonstrated that optimal PG expression was achieved with 2% methanol induction in BMMY, producing higher levels than those with ½ BSM. Among the transformants, PG.c2 produced the highest PG protein in BMMY medium induced with 2% methanol. Dot-blot analysis confirmed the secretion of PG, and LC-HRMS analysis demonstrated 100% amino acid sequence coverage of PG, confirming the integrity and completeness of the expressed protein. This study presented a newly modified PG gene cassette, inserted into pPICZαA vector, to express uniform secreted PG in P. pastoris GS115. By simplifying the precursor structure, a more homogeneous precursor product can be obtained, which is expected to simplify purification and also the downstream enzymatic process of PG into mature insulin glargine.
Co-Authors Abdjad Asih Nawangsih Abdjad Asih Nawangsih ABDUL MUNIF ABDUL MUNIF Akhmad Endang Zainal Hasan Alimuddin Alimuddin ALINA AKHDIYA Alina Akhdiya Andi Ernawati ANDINI PURNAWIJAYA Anja Meryandini Anja Meryandini Antonius Suwanto Ari Fina Bintarti Ari Fina Bintarti, Ari Fina ARI SUSILOWATI Aris Tjahjoleksono BRAMANTYO JATI PRASOJO Budi Tjahjono C Hanny Wijaya DIAH ISKANDRIATI Dina Aribah DINI NURDIANI Dudi Hardianto, Dudi Edi Husen EDI HUSEN Edi Husen EDI HUSEN Edi Husen Efrida Martius Engelhaupt, Martin ERNIN HIDAYATI Hamim Hamim Hari KAPLI Hermawaty Abubakar Iman Rusmana Irmawati Jun Nomura LAKSMI AMBARSARI Latifah Kosim Darusman Maggy Thenawidjaja Suhartono Maggy Thenawidjaya Suhartono Marini Wijayanti Meliah, Siti Mia Setiawati MONA PRIMANITA MUHAMMAD AGUS SUPRAYUDI Muhammad Faiz Amri Muhammad Zairin Jr MUNTI YUHANA Mutiha Panjaitan Nasution, Uli Julia Ni Putu Ratna Ayu Krishanti NI PUTU RATNA AYU KRISHANTI NISA RACHMANIA MUBARIK NURFITRIANI RINA Pamungkas, Joko Puspitasari, Esti Raden Ajie Syahbarie RAHAYU FITRIANI WANGSA PUTRIE RAHAYU WIDYASTUTI Rasti Saraswati RASTI SARASWATI Rasti Saraswati Rika Indri Astuti Rini, Adityawati Fajar Rury Eryna Putri Rustam, Yepy Hardi Sarah Asih Faulina Sarah Asih Faulina, Sarah Asih Satya, Andreas Adhi Sigit Tri Wibowo Sipriyadi Sipriyadi Sipriyadi Siska Tridesianti Siti Meliah Siti Sholekha Sri Budiarti Sri Budiarti Poerwanto sri murtini . Suryo Wiyono SUSILOWATI1 SUSILOWATI1 SYAMSUL BAHRI SYAMSUL BAHRI Tati Barus TEDJA IMAS Uci Cahlia Umi Fatmawati VINCENTIUS ARCA TESTAMENTI WAODE MUNAENI Wati, Cheppy WIDANARNI WIDANARNI Wiraswati, Sri Martina Yohanes Bernadino Putera Saju Yuli Siti Fatma YULIN LESTARI