scholarly journals Biochemical Characterization of a New β-Agarase from Cellulophaga Algicola

2019 ◽  
Vol 20 (9) ◽  
pp. 2143 ◽  
Author(s):  
Han ◽  
Zhang ◽  
Yang
Keyword(s):  
Structural Model ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Optimal Temperature ◽  
Salt Tolerant ◽  
Wide Ph Range ◽  
Specific Activities ◽  
Ph Range ◽  
Layer Chromatography

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

scholarly journals Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

2008 ◽  
Vol 75 (4) ◽  
pp. 1065-1073 ◽  
Author(s):  
Dolores Linde ◽  
Isabel Macias ◽  
Luc�a Fern�ndez-Arrojo ◽  
Francisco J. Plou ◽  
Antonio Jim�nez ◽  
...  
Keyword(s):  
Total Mass ◽  
Structural Model ◽  
Biochemical Characterization ◽  
Sucrose Solution ◽  
Catalytic Efficiency ◽  
Maximum Activity ◽  
Xanthophyllomyces Dendrorhous ◽  
Glycosyl Hydrolases ◽  
Mature Polypeptide

ABSTRACT An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70�C) and thermostability (with a T 50 in the range 66 to 71�C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k cat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter−1. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

scholarly journals Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 431
Author(s):  
Wenxin Wang ◽  
Jianxin Wang ◽  
Ruihua Yan ◽  
Runying Zeng ◽  
Yaqiang Zuo ◽  
...  
Keyword(s):  
Deep Sea ◽  
Maximum Activity ◽  
Cold Adapted ◽  
Working Temperature ◽  
Ph Values ◽  
Natural Substances ◽  
Ph Range ◽  
Layer Chromatography ◽  
Optimal Ph

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu2+ and Zn2+, whereas Fe2+ displayed an intensification of enzymatic activity. The Km and Vmax of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.

scholarly journals Localization and partial characterization of thermostable glucoamylase produced by newly isolated Thermomyces lanuginosus TO3 in submerged fermentation

2008 ◽  
Vol 51 (4) ◽  
pp. 657-665 ◽  
Author(s):  
Aline Zorzetto Lopes Gonçalves ◽  
Ana Flávia Azevedo Carvalho ◽  
Roberto da Silva ◽  
Eleni Gomes
Keyword(s):  
Submerged Fermentation ◽  
Sole Carbon Source ◽  
Soluble Starch ◽  
Maximum Activity ◽  
Thermomyces Lanuginosus ◽  
Fermentation Time ◽  
Enzyme Productivity ◽  
Wide Ph Range ◽  
Ph Range

Thermophilic Thermomyces lanuginosus strain TO3 was isolated from compost pile samples and was used for its ability to produce considerable glucoamylase activity when growing in liquid medium at 45ºC with starch as the sole carbon source. Enzyme productivity was high in submerged fermentation (SmF) with maximum activity of 13 U/mL after 168 h of fermentation. Higher quantities of glucose were released when the substrate for enzyme was soluble starch than maltose or maltooligosaccharides were used. The distribution of glucoamylase between the extracellular and cell-associated fractions varied according to fermentation time. Glucoamylase produced from T. lanuginosus TO3 had optimum activity at 65 ºC and good thermostability in the absence of substrate, with a half-life of 6 h at 60 ºC. The enzyme was stable over a wide pH range (4.0-10.0).

scholarly journals Exploring the Diversity of Fungal DyPs in Mangrove Soils to Produce and Characterize Novel Biocatalysts

2021 ◽  
Vol 7 (5) ◽  
pp. 321
Author(s):  
Amal Ben Ayed ◽  
Geoffroy Saint-Genis ◽  
Laurent Vallon ◽  
Dolores Linde ◽  
Annick Turbé-Doan ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Chemical Properties ◽  
Avicennia Marina ◽  
Reactive Black 5 ◽  
Wet Season ◽  
Rhizophora Stylosa ◽  
Genes Encoding ◽  
Wide Ph Range ◽  
Ph Range

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51–100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical–chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.

Biochemical and functional characterization of OsCSD3, a novel CuZn superoxide dismutase from rice

2018 ◽  
Vol 475 (19) ◽  
pp. 3105-3121 ◽  
Author(s):  
Ravi Prakash Sanyal ◽  
Amol Samant ◽  
Vishal Prashar ◽  
Hari Sharan Misra ◽  
Ajay Saini
Keyword(s):  
Oxidative Stress ◽  
Thermal Inactivation ◽  
Biochemical Characterization ◽  
Spectral Characteristics ◽  
Functional Characterization ◽  
Double Knockout ◽  
Wide Ph Range ◽  
Ph Range

Superoxide dismutases (SODs, EC 1.15.1.1) belong to an important group of antioxidant metalloenzymes. Multiple SODs exist for scavenging of reactive oxygen species (ROS) in different cellular compartments to maintain an intricate ROS balance. The present study deals with molecular and biochemical characterization of CuZn SOD encoded by LOC_Os03g11960 (referred to as OsCSD3), which is the least studied among the four rice isozymes. The OsCSD3 showed higher similarity to peroxisomal SODs in plants. The OsCSD3 transcript was up-regulated in response to salinity, drought, and oxidative stress. Full-length cDNA encoding OsCSD3 was cloned and expressed in Escherichia coli and analyzed for spectral characteristics. UV (ultraviolet)–visible spectroscopic analysis showed evidences of d–d transitions, while circular dichroism analysis indicated high β-sheet content in the protein. The OsCSD3 existed as homodimer (∼36 kDa) with both Cu2+ and Zn2+ metal cofactors and was substantially active over a wide pH range (7.0–10.8), with optimum pH of 9.0. The enzyme was sensitive to diethyldithiocarbamate but insensitive to sodium azide, which are the characteristics features of CuZn SODs. The enzyme also exhibited bicarbonate-dependent peroxidase activity. Unlike several other known CuZn SODs, OsCSD3 showed higher tolerance to hydrogen peroxide and thermal inactivation. Heterologous overexpression of OsCSD3 enhanced tolerance of E. coli sod double-knockout (ΔsodA ΔsodB) mutant and wild-type strain against methyl viologen-induced oxidative stress, indicating the in vivo function of this enzyme. The results show that the locus LOC_Os03g11960 of rice encodes a functional CuZn SOD with biochemical characteristics similar to the peroxisomal isozymes.

scholarly journals Overexpression and Biochemical Characterization of an Endo-α-1,4-polygalacturonase from Aspergillus nidulans in Pichia pastoris

2020 ◽  
Vol 21 (6) ◽  
pp. 2100
Author(s):  
Hua Xu ◽  
Pengfei Zhang ◽  
Yuchen Zhang ◽  
Zebin Liu ◽  
Xuebing Zhang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Aspergillus Nidulans ◽  
Biochemical Characterization ◽  
Structural Characteristics ◽  
Maximum Activity ◽  
Feed Additives ◽  
Moderate Activity ◽  
Multiple Sequence ◽  
Wide Ph Range ◽  
Ph Range

Pectinases have many applications in the industry of food, paper, and textiles, therefore finding novel polygalacturonases is required. Multiple sequence alignment and phylogenetic analysis of AnEPG (an endo-α-1,4-polygalacturonase from Aspergillus nidulans) and other GH 28 endo-polygalacturonases suggested that AnEPG is different from others. AnEPG overexpressed in Pichia pastoris was characterized. AnEPG showed the highest activity at pH 4.0, and exhibited moderate activity over a narrow pH range (pH 2.0–5.0) and superior stability in a wide pH range (pH 2.0–12.0). It displayed the highest activity at 60 °C, and retained >42.2% of maximum activity between 20 and 80 °C. It was stable below 40 °C and lost activity very quickly above 50 °C. Its apparent kinetic parameters against PGA (polygalacturonic acid) were determined, with the Km and kcat values of 8.3 mg/mL and 5640 μmol/min/mg, respectively. Ba2+ and Ni2+ enhanced activity by 12.2% and 9.4%, respectively, while Ca2+, Cu2+, and Mn2+ inhibited activity by 14.8%, 12.8%, and 10.2% separately. Analysis of hydrolysis products by AnEPG proved that AnEPG belongs to an endo-polygalacturonase. Modelled structure of AnEPG by I-TASSER showed structural characteristics of endo-polygalacturonases. This pectinase has great potential to be used in food industry and as feed additives.

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
Author(s):  
Dessy Natalia ◽  
Keni Vidilaseris ◽  
Pasjan Satrimafitrah ◽  
Wangsa Ismaya ◽  
Purkan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Saccharomycopsis Fibuligera ◽  
Sequence Identity ◽  
Ic50 Value ◽  
High Amino Acid ◽  
Ph Range

AbstractGlucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

scholarly journals Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

2021 ◽  
Author(s):  
Gregory S Bulmer ◽  
Fang Wei Yuen ◽  
Naimah Begum ◽  
Bethan S Jones ◽  
Sabine S Flitsch ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Enzyme Specificity ◽  
Fungal Enzymes ◽  
Glycoside Hydrolase Family 43 ◽  
Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

scholarly journals Characterization of a Robust and pH-Stable Tannase from Mangrove-Derived Yeast Rhodosporidium diobovatum Q95

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 546
Author(s):  
Jie Pan ◽  
Ni-Na Wang ◽  
Xue-Jing Yin ◽  
Xiao-Ling Liang ◽  
Zhi-Peng Wang
Keyword(s):  
Gallic Acid ◽  
Tannic Acid ◽  
Specific Activity ◽  
Maximum Activity ◽  
Sds Page ◽  
Rhodosporidium Diobovatum ◽  
Bacteria And Fungi ◽  
Ph Range ◽  
First Time

Tannase plays a crucial role in many fields, such as the pharmaceutical industry, beverage processing, and brewing. Although many tannases derived from bacteria and fungi have been thoroughly studied, those with good pH stabilities are still less reported. In this work, a mangrove-derived yeast strain Rhodosporidium diobovatum Q95, capable of efficiently degrading tannin, was screened to induce tannase, which exhibited an activity of up to 26.4 U/mL after 48 h cultivation in the presence of 15 g/L tannic acid. The tannase coding gene TANRD was cloned and expressed in Yarrowia lipolytica. The activity of recombinant tannase (named TanRd) was as high as 27.3 U/mL. TanRd was purified by chromatography and analysed by SDS-PAGE, showing a molecular weight of 75.1 kDa. The specific activity of TanRd towards tannic acid was 676.4 U/mg. Its highest activity was obtained at 40 °C, with more than 70% of the activity observed at 25–60 °C. Furthermore, it possessed at least 60% of the activity in a broad pH range of 2.5–6.5. Notably, TanRd was excellently stable at a pH range from 3.0 to 8.0; over 65% of its maximum activity remained after incubation. Besides, the broad substrate specificity of TanRd to esters of gallic acid has attracted wide attention. In view of the above, tannase resources were developed from mangrove-derived yeasts for the first time in this study. This tannase can become a promising material in tannin biodegradation and gallic acid production.

scholarly journals ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF BRUSH BORDERS FROM RABBIT KIDNEY

1970 ◽  
Vol 47 (3) ◽  
pp. 637-645 ◽  
Author(s):  
Sosamma J. Berger ◽  
Bertram Sacktor
Keyword(s):  
Brush Border ◽  
Renal Cortex ◽  
Biochemical Characterization ◽  
Rabbit Kidney ◽  
Renal Tubules ◽  
Marker Enzymes ◽  
Alkaline Phosphatases ◽  
Brush Borders ◽  
Specific Activities

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated. The procedure was monitored by phase and electron microscopy and marker enzymes, i.e. ATP:NMN adenylyl transferase, nuclear; cytochrome oxidase, mitochondrial; ß-glucuronidase, lysosomal; and glucose-6-Pase, microsomal; and indicated an essentially pure preparation of brush borders. The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders. Maltase was also found; the specific activities of the two enzymes in the brush borders were increased 10- to 20-fold. Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent. It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na+ + K+) ATPase, an oligomycin-insensitive Mg++ ATPase, and a Ca++-activated ATPase. Alkaline phosphatases, dephosphorylating ß-glycero-P, and trehalose-6-P were also present. The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations; however, activities were found in other subcellular fractions of the renal cortex. Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions. Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex.

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