scholarly journals Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

2020 ◽  
Vol 85 (1) ◽  
pp. 25-35
Author(s):  
Ana Balaz ◽  
Marija Blazic ◽  
Nikolina Popovic ◽  
Olivera Prodanovic ◽  
Raluca Ostafe ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phanerochaete Chrysosporium ◽  
Sodium Dodecyl ◽  
Cellobiose Dehydrogenase ◽  
Yeast Cells ◽  
Soluble Form ◽  
Kinetic Characterization ◽  
Methanol Induction ◽  
Ph Optimum ◽  
Mutant Proteins

Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZ? plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.

Methanol Induction Optimization for Recombinant Human Consensus Interferon Mutant Production in Pichia pastoris

2010 ◽  
Vol 150 ◽  
pp. 540-540
Author(s):  
Dan Wu ◽  
Ju Chu ◽  
Yu-You Hao ◽  
Yong-Hong Wang ◽  
Ying-Ping Zhuang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Induction ◽  
Consensus Interferon

Acetylsalicylic acid hydrolase of gastric mucosa.

1978 ◽  
Vol 234 (6) ◽  
pp. E606
Author(s):  
J G Spenney
Keyword(s):  
Gastric Mucosa ◽  
Enzymatic Activity ◽  
Acetylsalicylic Acid ◽  
Sodium Dodecyl ◽  
Sodium Cholate ◽  
Acid Hydrolase ◽  
Sulfhydryl Reagents ◽  
Ph Optimum ◽  
Diisopropyl Fluorophosphate

Acetylsalicylic acid hydrolase activity of rabbit fundic gastric mucosa has been isolated from the soluble 100,000 X g supernate. The enzymatic activity was partially purified by ammonium sulfate precipitation. The Km for acetylsalicylate was 2 mM and pH optimum was 8.6. The activity was insensitive to ionic strength, slightly inhibited by inclusion of 100 mM Cl-, and demonstrated no requirement for Ca2+ or Mg2+. Acetylsalicylic acid esterase was markedly inhibited by sodium cholate and sodium dodecyl sulfate. The enzyme was insensitive to sulfhydryl reagents with the exception of p-chloromercuribenzenesulfonic acid, which markedly inhibited the enzyme. Diisopropyl fluorophosphate (DFP) inhibited enzymatic activity with a Ki of 9 X 10(-9)M. Eserine was also inhibitory with a Ki of 0.25 mM. Inhibition by DFP at low concentration and by eserine at millimolar concentrations suggests that this enzyme is related to the group of aliphatic esterases. Identification of potent inhibitors will enable studies to define the role of this enzyme with the use of experimental preparations in which systemic toxicity can be avoided.

scholarly journals In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

2006 ◽  
Vol 72 (2) ◽  
pp. 1507-1514 ◽  
Author(s):  
Mark J. Daniels ◽  
Malcolm R. Wood ◽  
Mark Yeager
Keyword(s):  
Pichia Pastoris ◽  
Linear Relationship ◽  
Osmotic Shock ◽  
Water Channel ◽  
Yeast Cells ◽  
Mercury Chloride ◽  
Functional Assay ◽  
Channel Activity ◽  
Wild Type

ABSTRACT The water channel protein PvTIP3;1 (α-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

Differentiation of Yeast Cultures in Ruminant Feedingstuffs Using a Rapid DNA Fingerprinting Technique

1994 ◽  
Vol 1994 ◽  
pp. 101-101
Author(s):  
Elizabeth Moore ◽  
Denis R. Headon
Keyword(s):  
Sodium Dodecyl ◽  
Yeast Cells ◽  
Microbial Populations ◽  
Yeast Culture ◽  
Yeast Cultures ◽  
Yeast Strains ◽  
Genetic Sequences ◽  
Polymerase Chain ◽  
Different Strains ◽  
Triton X

Research indicates that certain yeast strains are beneficial in their capacity to stimulate key microbial populations. This stimulation is strain specific with similar yeast strains exerting their effect on totally different microbial populations. Future yeast culture supplements may contain mixtures of different strains designed to suit specific diets. This, therefore, requires the development of a rapid sensitive technique to differentiate among taxonomically similar yeast strains in animal diets. This technique, termed the Randomly Amplified Polymorphic DNA (RAPD) assay, is based upon the use of randomly designed short polynucleotide primers to amplify genetic sequences from the DNA of the desired yeast strain. Our objective involves the development of this technique to distinguish between closely related yeast strains present in feed. The feed sample investigated was a standard cattle ration containing three strains of Saccharomyces cerevisiae (1026, 2045 and 2020) and Candida utilis 3001 at a concentration of 106 CFU/g respectively. Isolation of single colonies of yeast strains present was achieved by feed extraction in dilution buffer followed by plating a series of dilutions on rose-bengal agar. Thirty randomly selected colonies were cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) broth for 24 - 30 hours at 30°C. Genomic DNA was isolated from yeast cells by standard methods based on subjection of the cells to vortex mixing in the presence of glass beads, triton X-100, sodium dodecyl sulphate, phenol and chloroform. Isolated DNA from randomly selected colonies was amplified by Polymerase Chain Reaction (PCR) for 45 cycles of 1 min at 94°C, 1 min at 36°C and 1 min at 72°C using randomly designed 10 bp primers.

scholarly journals Improved Production ofAspergillus usamiiendo-β-1,4-Xylanase inPichia pastorisvia Combined Strategies

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jianrong Wang ◽  
Yangyuan Li ◽  
Danni Liu
Keyword(s):  
Pichia Pastoris ◽  
Cell Viability ◽  
Recombinant Proteins ◽  
Recombinant Strain ◽  
Xylanase Activity ◽  
Vitreoscilla Hemoglobin ◽  
Wild Type ◽  
Methanol Induction ◽  
Aspergillus Usamii ◽  
Induction Temperature

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

1986 ◽  
Vol 64 (12) ◽  
pp. 1288-1293 ◽  
Author(s):  
Josefa M. Alonso ◽  
Amando Garrido-Pertierra
Keyword(s):  
Pseudomonas Putida ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Catabolic Pathway ◽  
Ph Optimum ◽  
Semialdehyde Dehydrogenase ◽  
Standard Assay ◽  
Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

Purification of glucosylceramidase by affinity chromatography

1982 ◽  
Vol 60 (11) ◽  
pp. 1025-1031 ◽  
Author(s):  
P. M. Strasberg ◽  
J. A. Lowden ◽  
D. Mahuran
Keyword(s):  
Sodium Dodecyl ◽  
Sodium Taurocholate ◽  
Low Ph ◽  
Affinity Column ◽  
Specific Technique ◽  
Natural Substrate ◽  
Ph Optimum ◽  
Specific Affinity ◽  
Glycol Solution ◽  
Slab Gels

Glucosylceramide:β-glucosidase (glucocerebrosidase, EC 3.2.1.45) has been purified 12 900-fold from human placenta using a specific affinity column. The ligand, glucosyl sphingosine, prepared from glucocerebroside by alkaline hydrolysis, was attached to epoxy-activated Sepharose 6B. The enzyme was applied to the column in citrate–butanol or citrate – ethylene glycol solution at its pH optimum (5.6). No enzyme was bound in the presence of detergent. Glucocerebrosidase was eluted with citrate–taurocholate buffer at low pH or with citrate-taurocholate buffer containing D-gluconolactone at the pH optimum. Citrate–taurocholate solution alone at the pH optimum would not elute the enzyme. The enzyme hydrolyzed both the natural substrate, glucocerebroside, and the artificial substrate, 4-methylumbelliferyl glucopyranoside. Glucocerebrosidase migrated as a single band on 10% sodium dodecyl sulfate–polyacrylamide tube and (or) slab gels, corresponding to a molecular weight of 75 000. It also ran as a single zone of enzyme activity or protein on native gels, composed of 2.2% polyacrylamide – 0.4% agarose containing sodium taurocholate. This is the first reported use of this gel system for the examination of glucocerebrosidase. Overall recovery is 30%. The procedure represents a more rapid and specific technique for purification of glucocerebrosidase than those previously reported.

scholarly journals Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 810-817
Author(s):  
KJ Balazovich ◽  
JE Smolen ◽  
LA Boxer
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Dose Dependent

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

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