scholarly journals A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study

2019 ◽  
Vol 20 (3) ◽  
pp. 780 ◽  
Author(s):  
Wei Tang ◽  
Dongming Lan ◽  
Zexin Zhao ◽  
Shuang Li ◽  
Xiuting Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Fermentation Broth ◽  
Catalytic Triad ◽  
Esterification Reaction ◽  
Monoacylglycerol Lipase ◽  
His Tag ◽  
Km Value ◽  
Mutagenesis Study ◽  
Hydrolysis Activity

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

scholarly journals Molecular, structural and biochemical characterization of a novel recombinant chlorophyllase from cyanobacterium Oscillatoria acuminata PCC 6304

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Sitian Gu ◽  
Xiaojun Dai ◽  
Zhengjun Xu ◽  
Qiwen Niu ◽  
Jiang Jiang ◽  
...  
Keyword(s):  
Chlorophyll A ◽  
Biochemical Characterization ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Oil Refining ◽  
Chlorophyll B ◽  
Pheophytin A ◽  
Signal Peptide Sequence ◽  
His Tag

Abstract Background Chlorophyllase catalyzes the hydrolysis of chlorophyll and produces chlorophyllide and phytol. Cyanobacterial chlorophyllases are likely to be more highly heterologously expressed than plant chlorophyllases. A novel recombinant chlorophyllase from the cyanobacterium Oscillatoria acuminata PCC 6304 was successfully expressed in Escherichia coli BL21(DE3). Results The putative N-terminal 28-amino-acid signal peptide sequence of O. acuminata chlorophyllase (OaCLH) is essential for its activity, but may confer poor solubility on OaCLH. The C-terminal fusion of a 6 × His tag caused a partial loss of activity in recombinant OaCLH, but an N-terminal 6 × His tag did not destroy its activity. The optimal pH and temperature for recombinant OaCLH activity are 7.0 and 40 °C, respectively. Recombinant OaCLH has hydrolysis activities against chlorophyll a, chlorophyll b, bacteriochlorophyll a, and pheophytin a, but prefers chlorophyll b and chlorophyll a as substrates. The results of site-directed mutagenesis experiments indicated that the catalytic triad of OaCLH consists of Ser159, Asp226, and His258. Conclusions The high-level expression and broad substrate specificity of recombinant OaCLH make it suitable for genetically engineering and a promising biocatalyst for industrial production, with applications in vegetable oil refining and laundry detergents.

scholarly journals Biochemical Characterization and Degradation Pattern of a Novel Endo-Type Bifunctional Alginate Lyase AlyA from Marine Bacterium Isoptericola halotolerans

Marine Drugs ◽  
2018 ◽  
Vol 16 (8) ◽  
pp. 258 ◽  
Author(s):  
Benwei Zhu ◽  
Limin Ning ◽  
Yucui Jiang ◽  
Lin Ge
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Alginate Lyase ◽  
Ionization Mass ◽  
Broad Substrate Specificity ◽  
Action Mode ◽  
Degradation Pattern ◽  
Km Value ◽  
Alginate Lyases ◽  
Layer Chromatography

Alginate lyases are important tools to prepare oligosaccharides with various physiological activities by degrading alginate. Particularly, the bifunctional alginate lyase can efficiently hydrolyze the polysaccharide into oligosaccharides. Herein, we cloned and identified a novel bifunctional alginate lyase, AlyA, with a high activity and broad substrate specificity from bacterium Isoptericola halotolerans NJ-05 for oligosaccharides preparation. For further applications in industry, the enzyme has been characterized and its action mode has been also elucidated. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 °C. Additionally, it possessed a broad substrate specificity, showing high activities towards not only polyM (polyβ-d-mannuronate) (7658.63 U/mg), but also polyG (poly α-l-guluronate) (8643.29 U/mg). Furthermore, the Km value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). TLC (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) were used to study the action mode of the enzyme, showing that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of the alginate lyases, with a broad substrate specificity.

scholarly journals Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

2003 ◽  
Vol 185 (15) ◽  
pp. 4442-4449 ◽  
Author(s):  
Gregory M. Cook ◽  
Stefanie Keis ◽  
Hugh W. Morgan ◽  
Christoph von Ballmoos ◽  
Ulrich Matthey ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Atp Synthesis ◽  
Intrinsic Property ◽  
Protein Sequencing ◽  
Hydrolysis Activity

ABSTRACT We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ε and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

scholarly journals Biochemical Characterization of the DNA Substrate Specificity of Werner Syndrome Helicase

2002 ◽  
Vol 277 (26) ◽  
pp. 23236-23245 ◽  
Author(s):  
Robert M. Brosh ◽  
Juwaria Waheed ◽  
Joshua A. Sommers
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Werner Syndrome ◽  
Dna Substrate

scholarly journals Origin and evolution of transporter substrate specificity within the NPF family

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Morten Egevang Jørgensen ◽  
Deyang Xu ◽  
Christoph Crocoll ◽  
Heidi Asschenfeldt Ernst ◽  
David Ramírez ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Biosynthetic Pathways ◽  
Cyanogenic Glucosides ◽  
Evolutionary Path ◽  
Substrate Specificities ◽  
Origin And Evolution ◽  
Glucosinolate Biosynthesis ◽  
Phylogenetic Lineage

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.

scholarly journals Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

2005 ◽  
Vol 187 (20) ◽  
pp. 7072-7080 ◽  
Author(s):  
Rie Matsumi ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Substrate Specificity ◽  
Signal Peptide ◽  
Biochemical Characterization ◽  
Transmembrane Domain ◽  
Biochemical Properties ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Hyperthermophilic Archaeon ◽  
Signal Peptide Peptidase ◽  
Putative Signal Peptide

ABSTRACT We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppATk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (ΔN54SppATk) was found to be stable against autoproteolysis and was examined further. ΔN54SppATk exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. ΔN54SppATk displayed a Km of 240 ± 18 μM and a V max of 27.8 ± 0.7 μmol min−1 mg−1 towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80°C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, ΔN54SppATk was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of ΔN54SppATk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.

scholarly journals Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form

2015 ◽  
Vol 71 (10) ◽  
pp. 1247-1250 ◽  
Author(s):  
Chang Min Kim ◽  
Jae Young Choi ◽  
Jong Hwan Yoon ◽  
Hyun Ho Park
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Gtp Hydrolysis ◽  
X Ray ◽  
Cell Parameters ◽  
Gtp Binding ◽  
His Tag ◽  
Hydrolysis Activity ◽  
Constitutively Active

RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed inEscherichia coliwith an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.4 Å from a crystal belonging to space groupI4, with unit-cell parametersa = 74.11,b= 74.11,c= 149.44 Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).

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