scholarly journals Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

Catalysts ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 254
Author(s):  
Xinxi Gu ◽  
Haiqiang Lu ◽  
Wei Chen ◽  
Xiangchen Meng
Keyword(s):  
Glycoside Hydrolase ◽  
Hydrolytic Activity ◽  
Initial Activity ◽  
Calculated Molecular Weight ◽  
Enzyme Cocktail ◽  
Activity Loss ◽  
Ph Range ◽  
Hydrolysis Of ◽  
New Strategies

Aspergillus fumigatus HBFH5 is a thermophilic fungus which can efficiently degrade lignocellulose and which produces a variety of glycoside hydrolase. In the present study, a novel β-mannanase gene (AfMan5A) was expressed in Pichia pastoris and characterized. AfMan5A is composed of 373 amino acids residues, and has a calculated molecular weight of 40 kDa. It has been observed that the amino acid sequence of AfMan5A showed 74.4% homology with the ManBK from Aspergillus niger. In addition, the recombined AfMan5A exhibited optimal hydrolytic activity at 60 °C and pH 6.0. It had no activity loss after incubation for 1h at 60 °C, while 65% of the initial activity was observed after 1 h at 70 °C. Additionally, it maintained about 80% of its activity in the pH range from 3.0 to 9.0. When carob bean gum was used as the substrate, the Km and Vmax values of AfMan5A were 0.21 ± 0.05 mg·mL−1 and 15.22 ± 0.33 U mg−1·min−1, respectively. AfMan5A and AfSwol showed a strong synergistic interaction on galactomannan degradation, increasing the reduction of the sugars by up to 31%. Therefore, these findings contribute to new strategies for improving the hydrolysis of galactomannan using the enzyme cocktail.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

A novel endo-β-1,4-xylanase GH30 from Dickeya dadantii DCE-01: Clone, expression, characterization, and ramie biological degumming function

2017 ◽  
Vol 89 (4) ◽  
pp. 463-472 ◽  
Author(s):  
Ruijun Wang ◽  
Zhengchu Liu ◽  
Lifeng Cheng ◽  
Shengwen Duan ◽  
Xiangyuan Feng ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Ph Value ◽  
Hydrolytic Activity ◽  
Glycoside Hydrolase Family ◽  
Dickeya Dadantii ◽  
Beechwood Xylan ◽  
Expression Characterization ◽  
Hydrolysis Of ◽  
Hydrolase Family

Xylanase plays an important role in the hydrolysis of hemicellulose and has gained much attention in the field of biological degumming. The research for xylanases with cellulase-free and high activity for biological degumming has intensified in recent years. In the present research, heterologous expression of a novel endo-β-1,4-xylanase (GH30) from Dickeya dadantii DCE-01 in Escherichia coli BL21 (DE3) was reported. Biochemical characterization of the enzyme and a potential application in ramie biological degumming was discussed. The results showed that the xylanase gene consists of 1251 nucleotides, belonging to glycoside hydrolase family 30 (GH30). The optimal activity of the xylanase was observed at 50℃ and a pH value of 6.4. The Km and Vmax values for beechwood xylan were 14.25 mg/mL and 296.6 μmol/mg, respectively. The catalytic activity was enhanced by addition of 1 mM Cu2+, Ca2+, Mg2+, and K+. The recombinant enzyme was specific for xylan substrates. The enzyme exhibited hydrolytic activity toward ramie hemicellulose. The recombinant xylanase could be effectively applied to ramie degumming.

scholarly journals Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Laura Plaza-Vinuesa ◽  
Oswaldo Hernandez-Hernandez ◽  
F. Javier Moreno ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Glycoside Hydrolase ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Structural Features ◽  
Biochemical Properties ◽  
Glycoside Hydrolase Family ◽  
Reaction Conditions ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Background α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. Results In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. Conclusions The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

scholarly journals Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

Marine Drugs ◽  
2020 ◽  
Vol 18 (6) ◽  
pp. 312 ◽  
Author(s):  
Dandan Li ◽  
Shangyong Li ◽  
Yanhong Wu ◽  
Mengfei Jin ◽  
Yu Zhou ◽  
...  
Keyword(s):  
Dairy Products ◽  
Marine Bacterium ◽  
Intestinal Flora ◽  
Lactose Hydrolysis ◽  
Glycoside Hydrolase Family ◽  
Transglycosylation Activity ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Hydrolase Family

As prebiotics, galacto-oligosaccharides (GOSs) can improve the intestinal flora and have important applications in medicine. β-galactosidases could promote the synthesis of GOSs in lactose and catalyze the hydrolysis of lactose. In this study, a new β-galactosidase gene (gal2A), which belongs to the glycoside hydrolase family 2, was cloned from marine bacterium Alteromonas sp. QD01 and expressed in Escherichia coli. The molecular weight of Gal2A was 117.07 kDa. The optimal pH and temperature of Gal2A were 8.0 and 40 °C, respectively. At the same time, Gal2A showed wide pH stability in the pH range of 6.0–9.5, which is suitable for lactose hydrolysis in milk. Most metal ions promoted the activity of Gal2A, especially Mn2+ and Mg2+. Importantly, Gal2A exhibited high transglycosylation activity, which can catalyze the formation of GOS from milk and lactose. These characteristics indicated that Gal2A may be ideal for producing GOSs and lactose-reducing dairy products.

scholarly journals Production and Characterization of Highly Thermostableβ-Glucosidase during the Biodegradation of Methyl Cellulose byFusarium oxysporum

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Folasade M. Olajuyigbe ◽  
Chidinma M. Nlekerem ◽  
Olusola A. Ogunyewo
Keyword(s):  
Residual Activity ◽  
Methyl Cellulose ◽  
Industrial Applications ◽  
Biofuel Production ◽  
Fermentable Sugars ◽  
Original Activity ◽  
Fermentation Period ◽  
Ph Range ◽  
Hydrolysis Of

Production ofβ-glucosidase fromFusarium oxysporumwas investigated during degradation of some cellulosic substrates (Avicel,α-cellulose, carboxymethyl cellulose (CMC), and methylcellulose). Optimized production ofβ-glucosidase using the cellulosic substrate that supported highest yield of enzyme was examined over 192 h fermentation period and varied pH of 3.0–11.0. Theβ-glucosidase produced was characterized for its suitability for industrial application. Methyl cellulose supported the highest yield ofβ-glucosidase (177.5 U/mg) at pH 6.0 and 30°C at 96 h of fermentation with liberation of 2.121 μmol/mL glucose. The crude enzyme had optimum activity at pH 5.0 and 70°C. The enzyme was stable over broad pH range of 4.0–7.0 with relative residual activity above 60% after 180 min of incubation.β-glucosidase demonstrated high thermostability with 83% of its original activity retained at 70°C after 180 min of incubation. The activity ofβ-glucosidase was enhanced by Mn2+and Fe2+with relative activities of 167.67% and 205.56%, respectively, at 5 mM and 360% and 315%, respectively, at 10 mM. The properties shown byβ-glucosidase suggest suitability of the enzyme for industrial applications in the improvement of hydrolysis of cellulosic compounds into fermentable sugars that can be used in energy generation and biofuel production.

scholarly journals Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina

1996 ◽  
Vol 318 (2) ◽  
pp. 711-716 ◽  
Author(s):  
Marco PICCININI ◽  
Adalberto MERIGHI ◽  
Renato BRUNO ◽  
Paolo CASCIO ◽  
Magda CURTO ◽  
...  
Keyword(s):  
Gene Product ◽  
Primary Structure ◽  
Protein Gene ◽  
Affinity Purification ◽  
Hydrolytic Activity ◽  
Protein Gene Product 9.5 ◽  
Protein Gene Product ◽  
Km Value ◽  
Ph Range

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin–Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.

Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell

1994 ◽  
Vol 72 (3-4) ◽  
pp. 157-162 ◽  
Author(s):  
Makoto Arao ◽  
Toru Yamaguchi ◽  
Toshitsugu Sugimoto ◽  
Masaaki Fukase ◽  
Kazuo Chihara
Keyword(s):  
Parathyroid Hormone ◽  
Molecular Mass ◽  
Hydrolytic Activity ◽  
High Molecular Mass ◽  
Phenylmethylsulfonyl Fluoride ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cell ◽  
Hydrolysis Of

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Sue, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from α-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250–300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1–84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.Key words: opossum kidney, parathyroid hormone, chymotrypsin, endopeptidase.

scholarly journals Characterization of an Alkaline Alginate Lyase with pH-Stable and Thermo-Tolerance Property

Marine Drugs ◽  
2019 ◽  
Vol 17 (5) ◽  
pp. 308 ◽  
Author(s):  
Yanan Wang ◽  
Xuehong Chen ◽  
Xiaolin Bi ◽  
Yining Ren ◽  
Qi Han ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Specific Activity ◽  
Alginate Lyase ◽  
Initial Activity ◽  
Alginate Oligosaccharides ◽  
Wide Ph Range ◽  
Encoding Gene ◽  
Ph Range ◽  
Alginate Lyases

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 °C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0–10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.

scholarly journals Characterization of the Family I inorganic pyrophosphatase fromPyrococcus horikoshiiOT3

Archaea ◽  
2005 ◽  
Vol 1 (6) ◽  
pp. 385-389 ◽  
Author(s):  
Sung-Jong Jeon ◽  
Kazuhiko Ishikawa
Keyword(s):  
Maximum Velocity ◽  
Heat Stability ◽  
Hydrolytic Activity ◽  
Heat Inactivation ◽  
Inorganic Pyrophosphatase ◽  
Gene Encoding ◽  
Sds Page ◽  
The Family ◽  
Hydrolysis Of

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeonPyrococcus horikoshiiOT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) fromP. horikoshiishowed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase fromP. horikoshii(PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability ofPhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+being most effective; Zn2+, Co2+and Mn2+efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Kmfor pyrophosphate and Mg2+were 113 and 303 µM, respectively; and maximum velocity,Vmax, was estimated at 930 U mg–1.

scholarly journals A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis

2019 ◽  
Vol 20 (9) ◽  
pp. 2261 ◽  
Author(s):  
Aline Vianna Bernardi ◽  
Deborah Kimie Yonamine ◽  
Sergio Akira Uyemura ◽  
Taisa Magnani Dinamarco
Keyword(s):  
Pichia Pastoris ◽  
Aspergillus Fumigatus ◽  
Lignocellulosic Biomass ◽  
Renewable Energy Sources ◽  
Industrial Applications ◽  
The Novel ◽  
Biomass Hydrolysis ◽  
Ph Range ◽  
Hydrolysis Of

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters Km and Vmax were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0–8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1–1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse “in natura”, sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.

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