scholarly journals Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase fromGeobacillussp. CHB1

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xianbo Jia ◽  
Jichen Chen ◽  
Chenqiang Lin ◽  
Xinjian Lin
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Residual Activity ◽  
Industrial Applications ◽  
Activity Level ◽  
High Yield ◽  
E Coli ◽  
Wide Range ◽  
Clone And Express

Catalases are widely used in many scientific areas. A catalase gene (Kat) fromGeobacillussp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed inEscherichia coli(E. coli), which was the first time to clone and express this type of catalase ofgenus Geobacillusstrains as far as we know. ThisKatgene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studiedBacillussp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble inE. coliand made up 30% of the totalE. coliprotein. Fermentation broth of the recombinantE. colishowed a high catalase activity level up to 35,831 U/mL which was only lower than recombinantBacillussp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg andKmof 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

scholarly journals AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB

2015 ◽  
Vol 81 (6) ◽  
pp. 1977-1987 ◽  
Author(s):  
Zhoukun Li ◽  
Jiale Wu ◽  
Biying Zhang ◽  
Fei Wang ◽  
Xianfeng Ye ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Specific Activity ◽  
Peptide Mass Fingerprinting ◽  
Soluble Starch ◽  
Industrial Applications ◽  
Product Analysis ◽  
Content Type ◽  
E Coli ◽  
Peptide Mass ◽  
Wide Range

ABSTRACTA novel α-amylase, AmyM, was purified from the culture supernatant ofCorallococcussp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence ofCorallococcuscoralloidesDSM 2259, the AmyM gene was identified and cloned intoEscherichia coli.amyMencodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases.amyMwas expressed inE. coliBL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM inE. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. TheKmandVmaxof recombinant AmyM for soluble starch were 6.61 mg ml−1and 44,301.5 μmol min−1mg−1, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.

scholarly journals Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain

Bioimpacts ◽  
2018 ◽  
Vol 9 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Azam Safary ◽  
Rezvan Moniri ◽  
Maryam Hamzeh-Mivehroud ◽  
Siavoush Dastmalchi
Keyword(s):  
Lymphoblastic Leukemia ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Substrate Affinity ◽  
Bacillus Sp ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Bacillus Strain ◽  
Antineoplastic Effect ◽  
Wide Range

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Biaosheng Lin ◽  
Zhijuan Li ◽  
Huixia Zhang ◽  
Jiangwen Wu ◽  
Maochun Luo
Keyword(s):  
Bacillus Subtilis ◽  
Fermentation Broth ◽  
Recombinant Expression ◽  
Product Yield ◽  
Industrial Applications ◽  
Cloning And Expression ◽  
Polyglutamic Acid ◽  
Recombinant Bacteria ◽  
Expression Product ◽  
Clone And Express

To clone and express theγ-polyglutamic acid (γ-PGA) synthetase genepgsBCA inBacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA intoBacillus subtilisWB600.PgsBCAwas expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesizeγ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher.γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates ofγ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of theγ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing theγ-PGA synthetase genepgsBCAinB. subtilissystem has potential industrial applications.

scholarly journals Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35

1999 ◽  
Vol 340 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Andrew BINIEDA ◽  
Martin FUHRMANN ◽  
Bruno LEHNER ◽  
Claudine REY-BERTHOD ◽  
Séverine FRUTIGER-HUGHES ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequence ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Active Enzyme ◽  
Gene Probe ◽  
Pimelic Acid ◽  
Pseudomonas Mendocina ◽  
Ph Optimum ◽  
E Coli

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of ≈ 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of ≈ 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, imelic cid-tilizing ) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

scholarly journals Dye Decoloring Peroxidase Structure, Catalytic Properties and Applications: Current Advancement and Futurity

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 955
Author(s):  
Lingxia Xu ◽  
Jianzhong Sun ◽  
Majjid A. Qaria ◽  
Lu Gao ◽  
Daochen Zhu
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
High Efficiency ◽  
Chemical Properties ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Research Strategy ◽  
Amino Acid Sequences ◽  
Alkali Resistance ◽  
Wide Range

Dye decoloring peroxidases (DyPs) were named after their high efficiency to decolorize and degrade a wide range of dyes. DyPs are a type of heme peroxidase and are quite different from known heme peroxidases in terms of amino acid sequences, protein structure, catalytic residues, and physical and chemical properties. DyPs oxidize polycyclic dyes and phenolic compounds. Thus they find high application potentials in dealing with environmental problems. The structure and catalytic characteristics of DyPs of different families from the amino acid sequence, protein structure, and enzymatic properties, and analyzes the high-efficiency degradation ability of some DyPs in dye and lignin degradation, which vary greatly among DyPs classes. In addition, application prospects of DyPs in biomedicine and other fields are also discussed briefly. At the same time, the research strategy based on genetic engineering and synthetic biology in improving the stability and catalytic activity of DyPs are summarized along with the important industrial applications of DyPs and associated challenges. Moreover, according to the current research findings, bringing DyPs to the industrial level may require improving the catalytic efficiency of DyP, increasing production, and enhancing alkali resistance and toxicity.

scholarly journals Characterization of an Atypical Superoxide Dismutase from Sinorhizobium meliloti

1999 ◽  
Vol 181 (15) ◽  
pp. 4509-4516 ◽  
Author(s):  
Renata Santos ◽  
Stephane Bocquet ◽  
Alain Puppo ◽  
Danièle Touati
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Genomic Library ◽  
Specific Activity ◽  
Growth Conditions ◽  
Aerobic Bacterium ◽  
Degenerate Primers ◽  
E Coli ◽  
Acid Protein

ABSTRACT Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. ThesodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M r of 22,430 and pI of 5.8.S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetictac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H2O2. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl2 or Fe(NH4)2(SO4)2, demonstrating the cambialistic nature of the S. melilotiSOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H2O2. Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H2O2of this unusual Fe-type SOD.

scholarly journals Genetic, Phenotypic, and Commercial Characterization of an Almond Collection from Sardinia

Plants ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 86 ◽  
Author(s):  
Emma Rapposelli ◽  
Maria Rigoldi ◽  
Daniela Satta ◽  
Donatella Delpiano ◽  
Sara Secci ◽  
...  
Keyword(s):  
Global Climate ◽  
Industrial Applications ◽  
Yield Potential ◽  
High Yield ◽  
Phenotypic Traits ◽  
Industrial Processing ◽  
High Oleic ◽  
Global Climate Changes ◽  
Content Ratio ◽  
Wide Range

Background: Recent nutritional and medical studies have associated the regular consumption of almonds with a wide range of health benefits. As a consequence, kernel quality has become an important goal for breeding, considering not only the chemical composition conferring a specific organoleptic quality but also physical traits related to industrial processing. Methods: We characterized an almond collection from Sardinia through analysis of 13 morpho-physiological traits and eight essential oil profiles. The genetic structure of the collection was studied by analyzing the polymorphism of 11 simple sequence repeats (SSR). Results: Both commercial and phenotypic traits showed wide ranges of variation. Most genotypes were early flowering with low yield potential. Several genotypes showed moderate to high yield and very interesting oil compositions of kernels. Based on 11 SSR profiles and Bayesian clustering, the Sardinian cultivars were assigned to groups which were differentiated for several agronomic and commercial traits. Conclusions: Several cultivars showed a high kernel oil content and high oleic to linoleic content ratio. Based on morphological traits, we propose that some of the analyzed cultivars could be interesting for industrial applications. Finally, we highlight the importance of characterizing early blooming cultivars for sites which are experiencing a rise in mean temperatures due to the effects of global climate changes.

scholarly journals A new method for the rapid purification of both the membrane-bound and released forms of the variant surface glycoprotein from Trypanosoma brucei

1985 ◽  
Vol 230 (1) ◽  
pp. 195-202 ◽  
Author(s):  
D G Jackson ◽  
M J Owen ◽  
H P Voorheis
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Aqueous Salt Solution ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
High Yield ◽  
Dependent Manner ◽  
Wide Range ◽  
Membrane Bound ◽  
Rapid Purification

A simple new technique was developed for the rapid purification of either the membrane-bound or the released forms of the variant surface glycoprotein of Trypanosoma brucei in high yield. Whole cells were used as the source of the membrane-bound form, and the supernatant of benzyl alcohol-treated cells was used as the source of the released form. The technique was based on extraction of the acid-treated protein into chloroform/methanol, followed by selective re-partition into aqueous salt solution. The yield of purified protein was found to be dependent critically on a low pH during the extraction/re-partition stages. This finding and the ability to cycle the protein repeatedly through organic and aqueous phases in a strictly pH-dependent manner suggested that the protein could undergo fully reversible denaturation/renaturation only while in an extensively protonated form. The yield was independent of the polarity of the organic phase and the protein concentration over a wide range. After purification, both forms retain their ability to react with specific antibody raised against the authentic native protein purified by conventional means. The amino acid composition and the identity of the N-terminal amino acid was the same for both forms of the protein. In addition, both forms had blocked C-terminal residues. There were determined to be 1.13 × 10(7) copies of the variant surface glycoprotein per cell.

scholarly journals Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase

1988 ◽  
Vol 250 (2) ◽  
pp. 429-434 ◽  
Author(s):  
J S Nishimura ◽  
J Ybarra ◽  
T Mitchell ◽  
P M Horowitz
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Beta Subunit ◽  
Native Enzyme ◽  
Tryptophan Residue ◽  
Beta Subunits ◽  
Post Translational Modification ◽  
E Coli ◽  
Succinyl Coa Synthetase

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between ‘strong’ and ‘weak’. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.

Sign in / Sign up

Export Citation Format

Share Document