Cloning and Characterization of a New Chitosanase From a Deep-Sea Bacterium Serratia sp. QD07

Chitosanase is a significant chitosan-degrading enzyme involved in industrial applications, which forms chitooligosaccharides (COS) as reaction products that are known to have various biological activities. In this study, the gene csnS was cloned from a deep-sea bacterium Serratia sp. QD07, as well as over-expressed in Escherichia coli, which is a new chitosanase encoding gene. The recombinant strain was cultured in a 5 L fermenter, which yielded 324 U/mL chitosanases. After purification, CsnS is a cold-adapted enzyme with the highest activity at 60°C, showing 37.5% of the maximal activity at 0°C and 42.6% of the maximal activity at 10°C. It exhibited optimum activity at pH 5.8 and was stable at a pH range of 3.4–8.8. Additionally, CsnS exhibited an endo-type cleavage pattern and hydrolyzed chitosan polymers to yield disaccharides and trisaccharides as the primary reaction products. These results make CsnS a potential candidate for the industrial manufacture of COS.

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Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties

Marine Drugs ◽

10.3390/md18080416 ◽

2020 ◽

Vol 18 (8) ◽

pp. 416

Author(s):

Yan Ma ◽

Jie Li ◽

Xin-Yue Zhang ◽

Hao-Dong Ni ◽

Feng-Biao Wang ◽

...

Keyword(s):

Metal Ion ◽

Brown Seaweed ◽

Alginate Lyase ◽

Industrial Applications ◽

Alginate Oligosaccharides ◽

Potential Applications ◽

Pharmaceutical Industries ◽

Ph Range ◽

Alginate Lyases

Alginate lyases play an important role in alginate oligosaccharides (AOS) preparation and brown seaweed processing. Many extracellular alginate lyases have been characterized to develop efficient degradation tools needed for industrial applications. However, few studies focusing on intracellular alginate lyases have been conducted. In this work, a novel intracellular alkaline alginate lyase Alyw202 from Vibrio sp. W2 was cloned, expressed and characterized. Secretory expression was performed in a food-grade host, Yarrowia lipolytica. Recombinant Alyw202 with a molecular weight of approximately 38.3 kDa exhibited the highest activity at 45 °C and more than 60% of the activity in a broad pH range of 3.0 to 10.0. Furthermore, Alyw202 showed remarkable metal ion-tolerance, NaCl independence and the capacity of degrading alginate into oligosaccharides of DP2-DP4. Due to the unique pH-stable and high salt-tolerant properties, Alyw202 has potential applications in the food and pharmaceutical industries.

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

Biochemistry Research International ◽

10.1155/2016/3978124 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 29

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.

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Purification and characterization of a novel NADPH(NADH)-dependent glyoxylate reductase from spinach leaves. Comparison of immunological properties of leaf glyoxylate reductase and hydroxypyruvate reductase

Biochemical Journal ◽

10.1042/bj2390653 ◽

1986 ◽

Vol 239 (3) ◽

pp. 653-659 ◽

Cited By ~ 30

Author(s):

L A Kleczkowski ◽

D D Randall ◽

D G Blevins

Keyword(s):

High Specificity ◽

Maximal Activity ◽

Purification And Characterization ◽

Leaf Extracts ◽

Hydroxypyruvate Reductase ◽

Ph Range ◽

Glyoxylate Reductase ◽

Spinach Leaves

A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.

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

Marine Drugs ◽

10.3390/md17050308 ◽

2019 ◽

Vol 17 (5) ◽

pp. 308 ◽

Cited By ~ 12

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.

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Production, purification and characterization of an exo-polygalacturonase from Penicillium janthinellum sw09

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-3765201620150051 ◽

2016 ◽

Vol 88 (suppl 1) ◽

pp. 479-487 ◽

Cited By ~ 5

Author(s):

YUPING MA ◽

SIWEN SUN ◽

HUI HAO ◽

CHUNPING XU

Keyword(s):

Gel Filtration ◽

Maximal Activity ◽

Growth Conditions ◽

Purification And Characterization ◽

Penicillium Janthinellum ◽

Soil Isolate ◽

Sds Page ◽

The Stability ◽

Ph Range

ABSTRACT A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 μmol/ (mL•min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20092261 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2261 ◽

Cited By ~ 7

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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Identification and molecular characterization of a psychrophilic GH1 β-glucosidase from the subtropical soil microorganism Exiguobacterium sp. GXG2

AMB Express ◽

10.1186/s13568-019-0873-7 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Bangqiao Yin ◽

Hengsen Gu ◽

Xueyan Mo ◽

Yue Xu ◽

Bing Yan ◽

...

Keyword(s):

Metal Ion ◽

Functional Characterization ◽

Industrial Applications ◽

Biochemical Properties ◽

Screening Strategy ◽

Soil Microorganism ◽

Potential Candidate ◽

Cold Adapted ◽

Subtropical Soil

Abstract The products of bacterial β-glucosidases with favorable cold-adapted properties have industrial applications. A psychrophilic β-glucosidase gene named bglG from subtropical soil microorganism Exiguobacterium sp. GXG2 was isolated and characterized by function-based screening strategy. Results of multiple alignments showed that the derived protein BglG shared 45.7% identities with reviewed β-glucosidases in the UniProtKB/Swiss-Prot database. Functional characterization of the β-glucosidase BglG indicated that BglG was a 468 aa protein with a molecular weight of 53.2 kDa. The BglG showed the highest activity in pH 7.0 at 35 °C and exhibited consistently high levels of activity within low temperatures ranging from 5 to 35 °C. The BglG appeared to be a psychrophilic enzyme. The values of Km, Vmax, kcat, and kcat/Km of recombinant BglG toward ρNPG were 1.1 mM, 1.4 µg/mL/min, 12.7 s−1, and 11.5 mM/s, respectively. The specific enzyme activity of BglG was 12.14 U/mg. The metal ion of Ca2+ and Fe3+ could stimulate the activity of BglG, whereas Mn2+ inhibited the activity. The cold-adapted β-glucosidase BglG displayed remarkable biochemical properties, making it a potential candidate for future industrial applications.

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Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Marine Drugs ◽

10.3390/md18110562 ◽

2020 ◽

Vol 18 (11) ◽

pp. 562

Author(s):

Marlene Vuillemin ◽

Artem S. Silchenko ◽

Hang Thi Thuy Cao ◽

Maxim S. Kokoulin ◽

Vo Thi Dieu Trang ◽

...

Keyword(s):

Marine Bacterium ◽

Divalent Cations ◽

Recombinant Expression ◽

Biological Activities ◽

Functional Characterization ◽

Maximal Activity ◽

Brown Macroalgae ◽

Pharmaceutical Uses ◽

Enzymatic Depolymerization

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

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Cloning, Secretory Expression and Characterization of a Unique pH-Stable and Cold-Adapted Alginate Lyase

Marine Drugs ◽

10.3390/md18040189 ◽

2020 ◽

Vol 18 (4) ◽

pp. 189 ◽

Cited By ~ 4

Author(s):

Zhi-Peng Wang ◽

Min Cao ◽

Bing Li ◽

Xiao-Feng Ji ◽

Xin-Yue Zhang ◽

...

Keyword(s):

Specific Activity ◽

Brown Seaweed ◽

Alginate Lyase ◽

Industrial Applications ◽

Degree Of Polymerization ◽

Cold Adapted ◽

Alginate Oligosaccharide ◽

Ph Range ◽

Alginate Lyases

Cold-adapted alginate lyases have unique advantages for alginate oligosaccharide (AOS) preparation and brown seaweed processing. Robust and cold-adapted alginate lyases are urgently needed for industrial applications. In this study, a cold-adapted alginate lyase-producing strain Vibrio sp. W2 was screened. Then, the gene ALYW201 was cloned from Vibrio sp. W2 and expressed in a food-grade host, Yarrowia lipolytica. The secreted Alyw201 showed the activity of 64.2 U/mL, with a molecular weight of approximate 38.0 kDa, and a specific activity of 876.4 U/mg. Alyw201 performed the highest activity at 30 °C, and more than 80% activity at 25–40 °C. Furthermore, more than 70% of the activity was obtained in a broad pH range of 5.0–10.0. Alyw201 was also NaCl-independent and salt-tolerant. The degraded product was that of the oligosaccharides of DP (Degree of polymerization) 2–6. Due to its robustness and its unique pH-stable property, Alyw201 can be an efficient tool for industrial production.

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

Marine Drugs ◽

10.3390/md19080431 ◽

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.

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