scholarly journals Combination of Genetic Manipulation Improved Saccharomycopsis fibuligera α-Amylase Secretion by Pichia pastoris

2019 ◽  
Vol 19 (2) ◽  
pp. 305
Author(s):  
Shabarni Gaffar ◽  
Dessy Natalia ◽  
Toto Subroto ◽  
Oo Suprijana ◽  
Soetijoso Soemitro
Keyword(s):  
Pichia Pastoris ◽  
Signal Peptide ◽  
Genetic Manipulation ◽  
Gene Dosage ◽  
Signal Sequence ◽  
Amylase Secretion ◽  
Saccharomycopsis Fibuligera ◽  
Peptide Modification ◽  
Peptide Gene ◽  
Native Signal Peptide

This study assessed the combinations of genetic manipulation; signal peptide modification, gene dosage increment and co-expression of folding component, to increase Saccharomycopsis fibuligera R64 α-amylase (Sfamy) secretion in Pichia pastoris. Sfamy native signal peptide was replaced with modified signal peptide which contained 15 amino acid of mouse salivary α-amylase signal peptide fused to the pro-region of the signal peptide of Saccharomyces cerevisiae α-mating factor (α-MF). Increase in gene dosage was identified by screening for P. pastoris harboring multicopies of the Sfamy gene. Whereas, co-expression of folding component was done by addition of Protein Disulfide Isomerase (PDI). Expression plasmids harboring Sfamy containing modified signal sequence (pPICZA-MS-Sfamy) was used to transform P. pastoris GS115, and gene dosage increment was screened using zeocin. Effect of PDI co-expression on secretion levels of Sfamy was assessed by constructing the pPIC3.5K-Pdi1 plasmid and introducing into P. pastoris harboring multicopies of MS-Sfamy for expression of Sfamy. Signal peptide modification consequently increased Sfamy secretion by P. pastoris by 3.3-fold compared to native signal peptide. Gene dosage increment had improved Sfamy secretion by 11-fold in P. pastoris [MS-Sfamy] resistant to 2000 μg/mL zeocin, compared to P. pastoris harboring one copy of WT-Sfamy. Hence, PDI co-expression increased the secretion of Sfamy by 2-fold as compared without PDI co-expression. In summary, the combination of genetic manipulation successfully increased Sfamy secretion by 20-fold compared to P. pastoris harboring one copy of WT-Sfamy.

Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris

2013 ◽  
Vol 52 (3) ◽  
pp. 177-183 ◽  
Author(s):  
Ashok Kumar Prasanna Vadhana ◽  
Premsingh Samuel ◽  
Ronald M Berin ◽  
Jayachandran Krishna ◽  
Kavitha Kamatchi ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Signal Peptide ◽  
Candida Antarctica ◽  
Candida Antarctica Lipase B ◽  
Candida Antarctica Lipase ◽  
Lipase B ◽  
Native Signal Peptide

From secretion in Pichia pastoris to application in apple juice processing: Exo-polygalacturonase from Sporothrix schenckii 1099-18

2021 ◽  
Vol 28 ◽  
Author(s):  
Ersin Karataş ◽  
Ahmet Tülek ◽  
Mehmet Mervan Çakar ◽  
Faruk Tamtürk ◽  
Fatih Aktaş ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Apple Juice ◽  
Biochemical Characterization ◽  
Signal Sequence ◽  
Sporothrix Schenckii ◽  
Juice Clarification ◽  
Pectinolytic Enzyme ◽  
Pectinolytic Enzymes ◽  
Protein Expression And Purification ◽  
Wide Range

Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo-PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

scholarly journals Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Selfela Restu Adina ◽  
Antonius Suwanto ◽  
Anja Meryandini ◽  
Esti Puspitasari
Keyword(s):  
Fatty Acid ◽  
Pichia Pastoris ◽  
Specific Activity ◽  
Signal Sequence ◽  
Micrococcus Luteus ◽  
Acid Methyl Ester ◽  
Industrial Applications ◽  
Expression Level ◽  
Lipase Gene ◽  
Acidic Lipase

Abstract Background Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris. Results LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0–5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na+, K+, Ca2+, Mg2+, Mn+) decreased enzyme activity up to 43.1%, while Fe2+ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%. Conclusions This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

Efficiency and diversity of protein localization by random signal sequences

1990 ◽  
Vol 10 (6) ◽  
pp. 3163-3173
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Protein Localization ◽  
Random Signal ◽  
Perinuclear Region ◽  
Hybrid Protein ◽  
Random Sequences ◽  
Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

The confirmation of the DNA uptake signal sequence needed for genetic manipulation in Haemophilus parasuis

2014 ◽  
Vol 173 (3-4) ◽  
pp. 395-396 ◽  
Author(s):  
Luhua Zhang ◽  
Ying Li ◽  
Ke Dai ◽  
Yiping Wen ◽  
Xintian Wen ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Signal Sequence ◽  
Dna Uptake ◽  
Haemophilus Parasuis

scholarly journals Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer

2020 ◽  
Vol 217 (6) ◽  
Author(s):  
Ivana Hermanova ◽  
Patricia Zúñiga-García ◽  
Alfredo Caro-Maldonado ◽  
Sonia Fernandez-Ruiz ◽  
Fernando Salvador ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Metastatic Prostate Cancer ◽  
Genetic Manipulation ◽  
Gene Dosage ◽  
Residual Activity ◽  
Low Frequency ◽  
Experimental Models ◽  
Cellular Systems ◽  
Prostate Tumorigenesis ◽  
Cancer Pathogenesis

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

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