scholarly journals Enhancement of a protocol purifying T1 lipase through molecular approach

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5833
Author(s):  
Che Haznie Ayu Che Hussian ◽  
Raja Noor Zaliha Raja Abd. Rahman ◽  
Adam Leow Thean Chor ◽  
Abu Bakar Salleh ◽  
Mohd Shukuri Mohamad Ali
Keyword(s):  
Rational Design ◽  
Double Point ◽  
Specific Activity ◽  
Point Mutations ◽  
Fold Increase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Site Directed Mutagenesis ◽  
Glutathione S Transferase ◽  
Direct Separation

T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.

scholarly journals Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by Streptococcus milleri NMSCC 061

2000 ◽  
Vol 66 (1) ◽  
pp. 23-28 ◽  
Author(s):  
M. Beukes ◽  
G. Bierbaum ◽  
H.-G. Sahl ◽  
J. W. Hastings
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Antimicrobial Substances ◽  
Streptococcus Milleri ◽  
N Terminus

ABSTRACT Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH2 SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH2 SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active againstBacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.

scholarly journals Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui-Hui Su ◽  
Fei Peng ◽  
Pei Xu ◽  
Xiao-Ling Wu ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rational Design ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Value Added ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glucaric Acid ◽  
Triple Mutant ◽  
Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Isolation, in the intact state, of the pterin molybdenum cofactor from xanthine oxidase

1989 ◽  
Vol 263 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J Deistung ◽  
R C Bray
Keyword(s):  
Aqueous Solution ◽  
Ion Exchange ◽  
Xanthine Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Molybdenum Cofactor ◽  
Anaerobic Conditions ◽  
Intact State

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.

scholarly journals Partial purification of two lithocholic acid-binding proteins from rat liver 100000g supernatants

1977 ◽  
Vol 165 (3) ◽  
pp. 425-429 ◽  
Author(s):  
R C Strange ◽  
R Cramb ◽  
J D Hayes ◽  
I W Percy-Robb
Keyword(s):  
Ion Exchange ◽  
Binding Proteins ◽  
Lithocholic Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Anion Binding ◽  
Partial Purification ◽  
Transferase Activity ◽  
Glutathione S Transferase

1. The partial purification of two lithocholic acid-binding proteins from liver 100 000g supernatants is described. 2. Gel-filtration, (NH4)2SO4 fractionation, Ca3(PO4)2 fractionation and ion-exchange chromatography were used. 3. Both proteins exhibited glutathione S-transferase activity; one may be the non-specific anion-binding protein ligandin. 4. Glutathione S-transferase activity of one of the binding proteins was inhibited by lithocholic acid.

scholarly journals Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

2002 ◽  
Vol 68 (4) ◽  
pp. 1980-1987 ◽  
Author(s):  
Yolanda Sanz ◽  
Fidel Toldrá
Keyword(s):  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Sulfhydryl Group ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lactobacillus Sakei ◽  
C Terminus ◽  
Arginine Aminopeptidase

ABSTRACT An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu2+, Hg2+, and Zn2+) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The Km values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

scholarly journals Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4806
Author(s):  
Zhongyi Cheng ◽  
Yao Lan ◽  
Junling Guo ◽  
Dong Ma ◽  
Shijin Jiang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Rational Design ◽  
Nitrile Hydratase ◽  
Elevated Temperatures ◽  
Residual Activity ◽  
Fold Increase ◽  
Computational Design ◽  
Site Directed Mutagenesis ◽  
Multiple Point ◽  
Substrate Affinity

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

Notes: Separation of 13-and 9-Hydroperoxide Lyase Activities in Cotyledons of Cucumber Seedlings

1989 ◽  
Vol 44 (9-10) ◽  
pp. 883-885 ◽  
Author(s):  
Kenji Matsui ◽  
Yasushi Shibata ◽  
Tadahiko Kajiwara ◽  
Akikazu Hatanaka
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Hydroperoxide Lyase ◽  
Cucumber Seedlings ◽  
Sh Reagents ◽  
Lyase Activity ◽  
Hydroperoxylinoleic Acid

Abstract In cucumber cotyledons, both C6- and C9- aldehyde were formed via hydroperoxide (HPO) lyase activity. Because it has not been elucidated whether these activities are attributed to one enzyme which can cleave both 13-and 9-HPO or to two or more enzymes each of which specifically cleaves 13-or 9-HPO , an attempt to separate HPO lyase activity was done. Ion exchange chromatography separated this activity into two fractions, one of which specifically cleaved 13-hydroperoxylinoleic acid and the other specifically cleaved the 9-isomer. 13-HPO-specific activity was most active at pH 8.0 and 9-HPO-specific one was at pH 6.5. SH -reagents inhibited both the lyases but to different extents.

Purification and characterization of the extracellular proteinase ofPseudomonas fluorescensNCDO 2085

1986 ◽  
Vol 53 (3) ◽  
pp. 457-466 ◽  
Author(s):  
David J. Fairbairn ◽  
Barry A. Law
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Heat Stable ◽  
D Values

SUMMAEYPseudomonas fluorescensNCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from otherPseudomonasspp.

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