Contribution of N-glycosylation site to the enzyme stability of hydroxynitrile lyase from Passiflora edulis in the differential expression system

2016 ◽  
Vol 33 ◽  
pp. S96
Author(s):  
Aem Nuylert ◽  
Yuko Ishida ◽  
Yasuhisa Asano
Keyword(s):  
Differential Expression ◽  
Enzyme Stability ◽  
Expression System ◽  
Glycosylation Site ◽  
Passiflora Edulis ◽  
Hydroxynitrile Lyase

scholarly journals Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

2003 ◽  
Vol 69 (2) ◽  
pp. 987-995 ◽  
Author(s):  
Thomas Bulter ◽  
Miguel Alcalde ◽  
Volker Sieber ◽  
Peter Meinhold ◽  
Christian Schlachtbauer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Enzyme Stability ◽  
Expression System ◽  
Fold Increase ◽  
Total Activity ◽  
Functional Expression ◽  
Glycosylation Site ◽  
C Terminus ◽  
Improved Stability

ABSTRACT Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

1998 ◽  
Vol 64 (3) ◽  
pp. 1163-1165 ◽  
Author(s):  
Georges Feller ◽  
Olivier Le Bussy ◽  
Charles Gerday
Keyword(s):  
Escherichia Coli ◽  
Enzyme Stability ◽  
Expression System ◽  
Wild Strain ◽  
Culture Temperature ◽  
E Coli ◽  
System A ◽  
Irreversible Denaturation ◽  
The Antarctic ◽  
Folding State

ABSTRACT α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.

scholarly journals Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

2013 ◽  
Vol 79 (15) ◽  
pp. 4727-4733 ◽  
Author(s):  
Kathrin Emmi Scholz ◽  
Benita Kopka ◽  
Astrid Wirtz ◽  
Martina Pohl ◽  
Karl-Erich Jaeger ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fusion Proteins ◽  
Enzyme Stability ◽  
Enzyme Analysis ◽  
Phase System ◽  
Wild Type ◽  
Hydroxynitrile Lyase ◽  
Fluorescent Reporter

ABSTRACTHydroxynitrile lyase fromArabidopsis thaliana(AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescencein vivoandin vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-typeAtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thoroughin vitrocharacterization ofin vivo-employed fluorescent fusion proteins.

scholarly journals Cloning and Expression of Staphylococcus Simulans Lysostaphin Enzyme Gene in Bacillus Subtilis WB600

2020 ◽  
Author(s):  
Babak Elyasi Far ◽  
Mehran Ragheb ◽  
Reza Rahbar ◽  
Ladan Mafakher ◽  
Neda Yousefi Nojookambari ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Enzyme Stability ◽  
Expression System ◽  
Residual Activity ◽  
Maximum Activity ◽  
Cloning And Expression ◽  
Gene Fragment ◽  
Alkaline Lysis ◽  
Enzyme Gene ◽  
Staphylococcus Simulans

Abstract Background: Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system.Results: Plasmid pACK1 of S. simulans was extracted using alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using PstI and XbaІ enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into B. subtilis WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to an about 27-kDa r-lysostaphin. Protein content was estimated 91µg/ml by Bradford assay.Conclusions: The recombinant lysostaphin represented 90% of its maximum activity at 40℃ and displayed good thermostability by keeping about 80% of its maximum activity at 45℃. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40℃ and showed good stability at 40℃ for 16 h incubation.

Crystal structure of hydroxynitrile lyase from passion fruit, Passiflora edulis

2016 ◽  
Vol 33 ◽  
pp. S96
Author(s):  
Fumihiro Motojima ◽  
Aem Nuylert ◽  
Yasuhisa Asano
Keyword(s):  
Crystal Structure ◽  
Passion Fruit ◽  
Passiflora Edulis ◽  
Hydroxynitrile Lyase

scholarly journals Molecular Cloning, Bioinformatic Analysis, and Expression ofBombyx moriLebocin 5 Gene Related toBeauveria bassianaInfection

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Dingding Lü ◽  
Chengxiang Hou ◽  
Guangxing Qin ◽  
Kun Gao ◽  
Tian Chen ◽  
...  
Keyword(s):  
Immune Response ◽  
Fat Body ◽  
Sequence Similarity ◽  
Expression System ◽  
Bioinformatic Analysis ◽  
Glycosylation Site ◽  
Pcr Analysis ◽  
Silkworm Larvae ◽  
Reading Frame ◽  
Prokaryotic Expression System

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm,Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected byBeauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defendB. bassianainfection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

scholarly journals Immobilization of Arabidopsis thaliana Hydroxynitrile Lyase (AtHNL) on EziG Opal

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 899
Author(s):  
José Coloma ◽  
Tim Lugtenburg ◽  
Muhammad Afendi ◽  
Mattia Lazzarotto ◽  
Paula Bracco ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Reaction Time ◽  
Continuous Flow ◽  
Flow System ◽  
Enzyme Stability ◽  
Reaction Medium ◽  
Specific Rate ◽  
Hydroxynitrile Lyase ◽  
Continuous Flow System ◽  
Space Time Yield

Arabidopsis thaliana hydroxynitrile lyase (AtHNL) catalyzes the selective synthesis of (R)-cyanohydrins. This enzyme is unstable under acidic conditions, therefore its immobilization is necessary for the synthesis of enantiopure cyanohydrins. EziG Opal is a controlled porosity glass material for the immobilization of His-tagged enzymes. The immobilization of His6-tagged AtHNL on EziG Opal was optimized for higher enzyme stability and tested for the synthesis of (R)-mandelonitrile in batch and continuous flow systems. AtHNL-EziG Opal achieved 95% of conversion after 30 min of reaction time in batch and it was recycled up to eight times with a final conversion of 80% and excellent enantioselectivity. The EziG Opal carrier catalyzed the racemic background reaction; however, the high enantioselectivity observed in the recycling study demonstrated that this was efficiently suppressed by using citrate/phosphate buffer saturated methyl-tert-butylether (MTBE) pH 5 as reaction medium. The continuous flow system achieved 96% of conversion and excellent enantioselectivity at 0.1 mL min−1. Lower conversion and enantioselectivity were observed at higher flow rates. The specific rate of AtHNL-EziG Opal in flow was 0.26 mol h−1 genzyme−1 at 0.1 mL min−1 and 96% of conversion whereas in batch, the immobilized enzyme displayed a specific rate of 0.51 mol h−1 genzyme−1 after 30 min of reaction time at a similar level of conversion. However, in terms of productivity the continuous flow system proved to be almost four times more productive than the batch approach, displaying a space-time-yield (STY) of 690 molproduct h−1 L−1 genzyme−1 compared to 187 molproduct h−1 L−1 genzyme−1 achieved with the batch system.

scholarly journals Human thrombopoietin structure–function relationships: identification of functionally important residues

1998 ◽  
Vol 333 (3) ◽  
pp. 729-734 ◽  
Author(s):  
Alexandre JAGERSCHMIDT ◽  
Valérie FLEURY ◽  
Marielle ANGER-LEROY ◽  
Corinne THOMAS ◽  
Magali AGNEL ◽  
...  
Keyword(s):  
Biological Effects ◽  
Expression System ◽  
Receptor Activation ◽  
Glycosylation Site ◽  
Eukaryotic Expression ◽  
Site Directed Mutagenesis ◽  
Receptor Dimerization ◽  
Haematopoietic Growth Factor ◽  
Proliferation And Differentiation ◽  
Receptor Interactions

Thrombopoietin (TPO) is a haematopoietic growth factor responsible for megakaryocyte progenitor proliferation and differentiation. It belongs to the four-helix-bundle cytokine family and exerts its biological effects through binding to a specific receptor, c-Mpl. With the use of site-directed mutagenesis we have generated 20 TPO mutants. Each of the TPO mutants was produced in a eukaryotic expression system and the mutants' ability to induce the proliferation of factor-dependent c-Mpl-expressing megakaryoblastic M-O7e cells was compared with that of wild-type TPO. Among the mutations studied, 10 lead to a significant decrease in TPO bioactivity. Of these ten residues, three are located in helix A of the protein (Arg10, Lys14 and Arg17) and four in helix D (His133, Gln132, Lys138 and Phe141), indicating that in TPO, as in other cytokines, these two helices are important for functional cytokine/receptor interactions. Surprisingly, mutant Arg10 → Ala (R10A) lacked any proliferative activity, despite the fact that this mutation was recently reported to have no effect on TPO/c-Mpl binding in a TPO phage ELISA [Pearce, Potts, Presta, Bald, Fendly and Wells (1997) J. Biol. Chem. 272, 20595–20602]. The lack of M-O7e proliferation is probably due to an inability of R10A mutant to promote receptor dimerization and thus receptor activation. Moreover we found that the Arg10 and Arg17 residues of TPO seem to be specific determinants for TPO/c-Mpl recognition. We also demonstrate that the O-glycosylation site located at position 110 of TPO is not necessary for the bioactivity of the cytokine.

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