Improved Production of Recombinant Myrosinase in Pichia pastoris

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

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The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

Gene ◽

10.1016/j.gene.2013.01.062 ◽

2013 ◽

Vol 519 (2) ◽

pp. 311-317 ◽

Cited By ~ 64

Author(s):

Geoff P. Lin-Cereghino ◽

Carolyn M. Stark ◽

Daniel Kim ◽

Jennifer Chang ◽

Nadia Shaheen ◽

...

Keyword(s):

Pichia Pastoris ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Protein Expression ◽

Secretion Signal ◽

Mating Factor ◽

Factor Secretion

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Secretion of Pem-CMG, a Peptide in the CHH/MIH/GIH Family of Penaeus monodon, in Pichia pastoris Is Directed by Secretion Signal of the α-Mating Factor from Saccharomyces cerevisiae

BMB Reports ◽

10.5483/bmbrep.2002.35.5.476 ◽

2002 ◽

Vol 35 (5) ◽

pp. 476-481 ◽

Cited By ~ 4

Author(s):

Supattra Treerattrakool ◽

Lily Eurwilaichitr ◽

Apinunt Udomkit ◽

Sakol Panyim

Keyword(s):

Saccharomyces Cerevisiae ◽

Pichia Pastoris ◽

Penaeus Monodon ◽

Secretion Signal ◽

Mating Factor

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Cloning and Characterization of Gluconolactone Oxidase of Penicillium cyaneo-fulvum ATCC 10431 and Evaluation of Its Use for Production of d-Erythorbic Acid in Recombinant Pichia pastoris

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5503-5510.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5503-5510 ◽

Cited By ~ 14

Author(s):

Tuomas Salusjärvi ◽

Nisse Kalkkinen ◽

Andrei N. Miasnikov

Keyword(s):

Pichia Pastoris ◽

Glucose Oxidase ◽

Signal Sequence ◽

Acid Fermentation ◽

Reaction Conditions ◽

Secretion Signal ◽

Genes Encoding ◽

Erythorbic Acid ◽

Cloned Gene

ABSTRACT A d-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced. Peptide sequences were used to isolate a cDNA clone encoding the enzyme. The cloned gene (accession no. AY576053 ) exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases. Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO. No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis. The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter. In order to evaluate the suitability of purified GLO for production of d-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions. Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of d-erythorbic acid from d-glucono-δ-lactone or (in combination with glucose oxidase and catalase) from glucose. We also demonstrated the feasibility of glucose-d-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient d-erythorbic acid-producing hosts.

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

Protein and Peptide Letters ◽

10.2174/1871530321666210106110400 ◽

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.

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

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00155-w ◽

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.

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Optimisation of Recombinant Myrosinase Production in Pichia pastoris

International Journal of Molecular Sciences ◽

10.3390/ijms22073677 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3677

Author(s):

Zuzana Rosenbergová ◽

Kristína Kántorová ◽

Martin Šimkovič ◽

Albert Breier ◽

Martin Rebroš

Keyword(s):

Pichia Pastoris ◽

Plant Defence ◽

Plant Secondary Metabolites ◽

Specific Productivity ◽

Fermentation Conditions ◽

Threefold Increase ◽

Dry Cell Weight ◽

First Time ◽

Hydrolysis Of ◽

Dry Cell

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.

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Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1812-1819.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1812-1819

Author(s):

C N Chang ◽

M Matteucci ◽

L J Perry ◽

J J Wulf ◽

C Y Chen ◽

...

Keyword(s):

Amino Acid ◽

Signal Sequence ◽

Phosphoglycerate Kinase ◽

Yeast Cells ◽

Signal Peptidase ◽

Human Interferon ◽

Expression Plasmid ◽

Kinase Gene ◽

Secretion Signal ◽

Yeast Invertase

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.

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Cloning of fibA, Encoding an Immunogenic Subunit of the Fibril-Like Surface Structure ofPeptostreptococcus micros

Journal of Bacteriology ◽

10.1128/jb.181.8.2485-2491.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2485-2491 ◽

Cited By ~ 6

Author(s):

B. H. A. Kremer ◽

J. J. E. Bijlsma ◽

J. G. Kusters ◽

J. de Graaff ◽

T. J. M. van Steenbergen

Keyword(s):

Amino Acid ◽

Surface Structure ◽

Signal Sequence ◽

Open Reading Frames ◽

Periodontal Pathogen ◽

Immunogold Electron Microscopy ◽

Expression Library ◽

Gram Positive ◽

Secretion Signal ◽

Specific Serum

ABSTRACT Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogenPeptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a λTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designatedfibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.

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