scholarly journals Biochemical Properties of Pectate Lyases Produced by Three Different Bacillus Strains Isolated from Fermenting Cocoa Beans and Characterization of Their Cloned Genes

2010 ◽  
Vol 76 (15) ◽  
pp. 5214-5220 ◽  
Author(s):  
Honor� G. Ouattara ◽  
Sylvie Reverchon ◽  
S�bastien L. Niamke ◽  
William Nasser
Keyword(s):  
Specific Activity ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Cocoa Beans ◽  
Chromatography Method ◽  
Pectate Lyases ◽  
Pectinolytic Enzymes ◽  
One Step ◽  
Random Manner

ABSTRACT Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The purified enzymes exhibited optimum activity at 60�C, and the half-time of thermoinactivation at this temperature was approximately 30 min. Pel-22 had a low specific activity compared with the other two enzymes. However, it displayed high affinity for the substrate, about 2.5-fold higher than those of Pel-66 and Pel-90. The optimum pHs were 7.5 for Pel-22 and 8.0 for Pel-66 and Pel-90. The three enzymes trans-eliminated polygalacturonate in a random manner to generate two long oligogalacturonides, as well as trimers and dimers. A synergistic effect was observed between Pel-22 and Pel-66 and between Pel-22 and Pel-90, but not between Pel-90 and Pel-66. The Pels were also strongly active on highly methylated pectins (up to 60% for Pel-66 and Pel-90 and up to 75% for Pel-22). Fe2+ was found to be a better cofactor than Ca2+ for Pel-22 activity, while Ca2+ was the best cofactor for Pel-66 and Pel-90. The amino acid sequences deduced from the cloned genes showed the characteristics of Pels belonging to Family 1. The pel-66 and pel-90 genes appear to be very similar, but they are different from the pel-22 gene. The characterized enzymes form two groups, Pel-66/Pel-90 and Pel-22; members of the different groups might cooperate to depolymerize pectin during the fermentation of cocoa beans.

scholarly journals Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Peixian Bai ◽  
Liyuan Wang ◽  
Kang Wei ◽  
Li Ruan ◽  
Liyun Wu ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Camellia Sinensis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Sequences ◽  
Biochemical Properties ◽  
High Similarity ◽  
New Gene ◽  
Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

2018 ◽  
Vol 43 (6) ◽  
pp. 638-650
Author(s):  
Ruth Ololade Amiola ◽  
Adedeji Nelson Ademakinwa ◽  
Zainab Adenike Ayinla ◽  
Esther Nkechi Ezima ◽  
Femi Kayode Agboola
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Biochemical Properties ◽  
Subunit Molecular Weight ◽  
Cyanoalanine Synthase ◽  
Germinating Seeds ◽  
Sorghum Bicolor L

Abstract Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

scholarly journals Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene (pdc) and Comparison to Bacterial Homologues

2002 ◽  
Vol 68 (6) ◽  
pp. 2869-2876 ◽  
Author(s):  
Krishnan Chandra Raj ◽  
Lee A. Talarico ◽  
Lonnie O. Ingram ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Codon Usage ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Biochemical Properties ◽  
Kinetic Properties ◽  
E Coli ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Zymobacter Palmae

ABSTRACT Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein−1) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

scholarly journals Characterization of Polyphenols from Various Cocoa (Theobroma cacao L.) Clones During Fermentation

2018 ◽  
Vol 34 (2) ◽  
pp. 104-112
Author(s):  
Muhammad Isa Dwijatmoko ◽  
Budi Nurtama ◽  
Nancy Dewi Yuliana ◽  
Misnawi Misnawi
Keyword(s):  
Theobroma Cacao ◽  
Antioxidant Property ◽  
Cocoa Bean ◽  
Flavor Quality ◽  
Cocoa Beans ◽  
Total Polyphenols ◽  
One Step ◽  
Theobroma Cacao L

Cocoa bean is a rich source of polyphenols, which are the largest group secondary metabolite with natural antioxidant property. Polyphenols from cocoabeans was reported to possess health benefits. Fermentation, one step in cocoa processing is needed to improve the quality of cocoa in which the concentration of cocoa bean polyphenols might decrease significantly through oxidation and exudation. Cocoa polyphenols content among different cocoa clones mightalso vary. The aims of this study were to determine total polyphenols, total flavanoid, epicatechin, and catechin content in several cocoa clones, those wereSulawesi 1, Sulawesi 2, ICCRI 03, and KW 617. Until now, characterization of polyphenols from those clones has not been reported. The effect of five daysfermentation to those parameters was also studied. The results of the study showed that fermentation and type of clones significantly affected total of polyphenols, total of flavanoids, epicatechin, and catechin content of the cocoa, there is also an interaction between fermentation and type of clones. Unfermented of Sulawesi 1 had the highest total polyphenols of 96.94±5.83 mg/g, total flavanoids of 90.92±1.89 mg/g, epicatechin of 52.50±0.46 mg/g, and catechin of 1.99±0.02 mg/g content compared to other clones. Among five days fermented cocoa beans, Sulawesi 2 showed the highest total polyphenols and total flavanoids content, while ICCRI 03 had the highest epicatechin and catechin content than other clones. Thus, in can be concluded that although fermentation is required to improve the flavor quality of cocoa, it significantly reduced the content of bioactive compounds. This effect varied amongdifferent cocoa clones.

One step forward to a scalable synthesis of platinum–yttrium alloy nanoparticles on mesoporous carbon for the oxygen reduction reaction

2016 ◽  
Vol 4 (31) ◽  
pp. 12232-12240 ◽  
Author(s):  
R. Brandiele ◽  
C. Durante ◽  
E. Grądzka ◽  
G. A. Rizzi ◽  
J. Zheng ◽  
...  
Keyword(s):  
Oxygen Reduction Reaction ◽  
Mesoporous Carbon ◽  
Specific Activity ◽  
Reduction Reaction ◽  
High Mass ◽  
Synthesis And Characterization ◽  
Alloy Nanoparticles ◽  
One Step ◽  
Scalable Synthesis

In this paper, we report the synthesis and characterization of nanoparticles of a PtxY alloy supported on a commercial mesoporous carbon with high mass and specific activity.

Biochemical characterization of α- and β-glucosidases in alimentary canal, salivary glands and haemolymph of the rice green caterpillar, Naranga aenescens M. (Lepidoptera: Noctuidae)

Biologia ◽  
2012 ◽  
Vol 67 (6) ◽  
Author(s):  
Ameneh Asadi ◽  
Mohammad Ghadamyari ◽  
Reza Sajedi ◽  
Jalal Sendi ◽  
Mehrdad Tabari
Keyword(s):  
Salivary Gland ◽  
Salivary Glands ◽  
Alimentary Canal ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Lepidoptera Noctuidae ◽  
Optimal Ph

AbstractThe biochemical properties of α- and β-glucosidase in salivary glands, alimentary canal and haemolymph of Naranga aenescens larvae, one of the most damaging pests of the rice crop in Iran, were investigated. The specific activity of α-glucosidases were 3.88, 2.74 and 1.58 μmol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The specific activity of β-glucosidases were 1.27, 0.077 and 0.414 μmol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The optimal pH for α-glucosidases were 6.0, 6.0–8.0 and 6.0 and the maximum activity for β-glucosidases were obtained at pH 6.0, 5.0–7.0 and 5.0 in alimentary canal, salivary glands and haemolymph, respectively. The optimum temperatures for β-glucosidases were determined at 55°C in alimentary canal, 35–45°C in salivary glands and 55°C in haemolymph, whereas the α-glucosidases reached their optimum at 45°C in all three tissues. Effect of metal ions on the activity of α- and β-glucosidases showed that K+ (20 mM) and Mg2+ (10 and 20 mM) increased N. aenescens α- and β-glucosidases activities from salivary glands, while Ca2+ increased α- and β-glucosidases activities in haemolymph. In the presence of Fe2+, Mn2+, Hg+ and Zn2+ (10, 20 mM) and Hg2+ (20 mM), these enzymes from all tissues were completely inactivated. K m values were estimated for the α-glucosidases as 3.96, 0.547 and 3.084 mM and for β-glucosidases as 1.93, 1.014 and 1.93 mM in the alimentary canal, salivary gland and haemolymph, respectively. The zymogram analyses of N. aenescens crude extracts indicated the presence of at least two isoforms for α-glucosidase and one isoform for β-glucosidase.

scholarly journals Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties

2003 ◽  
Vol 376 (1) ◽  
pp. 261-268 ◽  
Author(s):  
Lourdes RODRIGO ◽  
Fernando GIL ◽  
Antonio F. HERNANDEZ ◽  
Olga LOPEZ ◽  
Antonio PLA
Keyword(s):  
Affinity Chromatography ◽  
Rat Liver ◽  
Specific Activity ◽  
Liver Microsomes ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Rabbit Serum ◽  
Rat Liver Microsomes ◽  
Apparent Homogeneity

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435–33442] and liver [Ozols (1999) Biochem. J. 338, 265–275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE–Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A–Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, Km and heat and pH stability.

scholarly journals Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering

2019 ◽  
Vol 20 (6) ◽  
pp. 1269 ◽  
Author(s):  
Sakda Yainoy ◽  
Thanawat Phuadraksa ◽  
Sineewanlaya Wichit ◽  
Maprang Sompoppokakul ◽  
Napat Songtawee ◽  
...  
Keyword(s):  
Rational Design ◽  
Specific Activity ◽  
Homology Modelling ◽  
Biochemical Properties ◽  
Wild Type ◽  
E Coli ◽  
Sequence Identity ◽  
High Expression Level ◽  
The Ideal

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at −20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

scholarly journals SignalP 6.0 achieves signal peptide prediction across all types using protein language models

2021 ◽  
Author(s):  
Felix Teufel ◽  
José Juan Almagro Armenteros ◽  
Alexander Rosenberg Johansen ◽  
Magnús Halldór Gislason ◽  
Silas Irby Pihl ◽  
...  
Keyword(s):  
Sequence Data ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Language Models ◽  
Metagenomic Data ◽  
Living Organisms ◽  
Peptide Prediction ◽  
Control Protein

Signal peptides (SPs) are short amino acid sequences that control protein secretion and translocation in all living organisms. As experimental characterization of SPs is costly, prediction algorithms are applied to predict them from sequence data. However, existing methods are unable to detect all known types of SPs. We introduce SignalP 6.0, the first model capable of detecting all five SP types. Additionally, the model accurately identifies the positions of regions within SPs, revealing the defining biochemical properties that underlie the function of SPs in vivo. Results show that SignalP 6.0 has improved prediction performance, and is the first model to be applicable to metagenomic data. SignalP 6.0 is available at https://services.healthtech.dtu.dk/service.php?SignalP-6.0

scholarly journals Characterization of the Exopolygalacturonate Lyase PelX of Erwinia chrysanthemi 3937

1999 ◽  
Vol 181 (5) ◽  
pp. 1652-1663 ◽  
Author(s):  
Vladimir E. Shevchik ◽  
Harry C. M. Kester ◽  
Jacques A. E. Benen ◽  
Jaap Visser ◽  
Janine Robert-Baudouy ◽  
...  
Keyword(s):  
Pectate Lyase ◽  
Erwinia Chrysanthemi ◽  
Receptor Protein ◽  
Polymeric Chain ◽  
Pectate Lyases ◽  
Pectinolytic Enzymes ◽  
Terminal Amino ◽  
Almost All ◽  
Binding Subsites

ABSTRACT Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-α-d-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pelgenes deleted, we cloned a pectinase gene identified aspelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved inpelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites −2 to +2. PelX and PehX were shown to be localized in the periplasm ofE. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

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