scholarly journals Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris

2002 ◽  
Vol 366 (2) ◽  
pp. 603-611 ◽  
Author(s):  
Guan-Chiun LEE ◽  
Li-Chiun LEE ◽  
Vasyl SAVA ◽  
Jei-Fu SHAW
Keyword(s):  
Pichia Pastoris ◽  
Biochemical Characterization ◽  
Candida Rugosa ◽  
Residual Activity ◽  
Site Directed Mutagenesis ◽  
Overlap Extension ◽  
Chain Lengths ◽  
Serine Codons ◽  
Long Chain ◽  
Lip2 Lipase

The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH7 and a broad temperature optimum in the range 30–50°C. The enzyme retained 80% of residual activity after being heated at 70°C for 10min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12–C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.

scholarly journals Biochemical characterization of the PHARC-associated serine hydrolase ABHD12 reveals its preference for very-long-chain lipids

2018 ◽  
Vol 293 (44) ◽  
pp. 16953-16963 ◽  
Author(s):  
Alaumy Joshi ◽  
Minhaj Shaikh ◽  
Shubham Singh ◽  
Abinaya Rajendran ◽  
Amol Mhetre ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Biochemical Characterization ◽  
Fatty Acyl ◽  
Endoplasmic Reticulum Membrane ◽  
Long Chain Fatty Acids ◽  
Serine Hydrolase ◽  
Brain Membrane ◽  
Chain Lengths ◽  
Long Chain

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.

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.

Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active

2013 ◽  
Vol 450 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Sweta Maheshwari ◽  
Marina Lavigne ◽  
Alicia Contet ◽  
Blandine Alberge ◽  
Emilie Pihan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cellular Localization ◽  
Biochemical Characterization ◽  
De Novo ◽  
Membrane Lipids ◽  
Polyclonal Antibodies ◽  
Homology Modelling ◽  
Site Directed Mutagenesis ◽  
Recombinant Enzymes ◽  
Parasite Life Cycle

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis–Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

scholarly journals Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1

2017 ◽  
Vol 30 (8) ◽  
pp. 571-571 ◽  
Author(s):  
Svetlana A Korban ◽  
Kirill S Bobrov ◽  
Maria A Maynskova ◽  
Stanislav N Naryzhny ◽  
Olga L Vlasova ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Heterologous Expression ◽  
Biochemical Characterization ◽  
Fusarium Proliferatum

The Use of N-alkanes for Estimating Intake and Passage Rate in Horses

1996 ◽  
Vol 1996 ◽  
pp. 98-98
Author(s):  
B M L McLean ◽  
R W Mayes ◽  
F D DeB Hovell
Keyword(s):  
Recent Work ◽  
Chain Length ◽  
Carbon Chain ◽  
Carbon Chain Length ◽  
Passage Rate ◽  
Forage Crops ◽  
Carbon Chains ◽  
Naturally Occurring ◽  
Chain Lengths ◽  
Long Chain

Alkanes occur naturally in all plants, although forage crops tend to have higher alkane contents than cereals. N-alkanes have odd-numbered carbon chains. They are ideal for use as markers in feed trials, because, they are inert, indigestible and naturally occurring, and can be recovered in animal faeces. Synthetic alkanes (even-numbered carbon chains) are available commercially and can also used as external markers. Dove and Mayes (1991) cite evidence indicating that faecal recovery of alkanes in ruminants increases with increasing carbon-chain length. Thus the alkane “pairs” (e.g. C35 & C36, and C32 & C33) are used in calculating intake and digestibility because they are long chain and adjacent to each other. However, recent work by Cuddeford and Mayes (unpublished) has found that in horses the faecal recovery rates are similar regardless of chain lengths.

Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands

2005 ◽  
Vol 434 (1) ◽  
pp. 201-211 ◽  
Author(s):  
Mirela Cristea ◽  
Åke Engström ◽  
Chao Su ◽  
Lena Hörnsten ◽  
Ernst H. Oliw
Keyword(s):  
Pichia Pastoris ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Metal Ligands

Antibacterial Effects of Long-Chain Polyphosphates on Selected Spoilage and Pathogenic Bacteria

2008 ◽  
Vol 71 (7) ◽  
pp. 1401-1405 ◽  
Author(s):  
JEREMY A. OBRITSCH ◽  
DOJIN RYU ◽  
LUCINA E. LAMPILA ◽  
LLOYD B. BULLERMAN
Keyword(s):  
Food Industry ◽  
Pathogenic Bacteria ◽  
Antimicrobial Activities ◽  
Lag Phase ◽  
E Coli ◽  
Inhibition Of Growth ◽  
K 12 ◽  
Chain Lengths ◽  
And Staphylococcus Aureus ◽  
Long Chain

The antimicrobial activities of four long-chain food-grade polyphosphates were studied at concentrations allowed in the food industry (<5,000 ppm) in defined basal media by determining the inhibition of growth of three gram-negative and four gram-positive spoilage and pathogenic bacteria. Both generation time and lag phase of Escherichia coli K-12, E. coli O157: H7, and Salmonella Typhimurium were increased with all of the polyphosphates tested. Bacillus subtilis and Staphylococcus aureus were more sensitive to polyphosphates, but not in all cases, with multiphased growth. The growth of Lactobacillus plantarum was inhibited by polyphosphates at concentrations above 750 ppm, but the lag time of Listeria monocytogenes was shortened by the presence of polyphosphates. No single polyphosphate was maximally inhibitory against all bacteria. Polyphosphates with chain lengths of 12 to 15 were significantly different from those with chain lengths of 18 to 21 depending on the organism and concentrations of polyphosphate used. Overall, higher polyphosphate concentrations resulted in greater inhibition of bacterial growth.

Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

2004 ◽  
Vol 37 (1) ◽  
pp. 61-71 ◽  
Author(s):  
Guillaume G Barbier ◽  
Rama C Joshi ◽  
Ellen R Campbell ◽  
Wilbur H (Bill) Campbell
Keyword(s):  
Pichia Pastoris ◽  
Nitrate Reductase ◽  
Biochemical Characterization
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