scholarly journals Evaluation of Cellulolytic Endo-1,4-β-D-Glucanase Activity in the Digestive Fluid of Adult Phytophagous Beetle Hoplasoma unicolor

2021 ◽  
Vol 32 (3) ◽  
pp. 53-68
Author(s):  
Mohammad Mosleh Uddin ◽  
Suzana Afrin Lima ◽  
Tanim Jabid Hossain ◽  
Newton Kar ◽  
Yeasmin Zahan ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Specific Activity ◽  
Agar Plate ◽  
Industrial Applications ◽  
Cellulolytic Activity ◽  
Glucanase Activity ◽  
Endoglucanase Activity ◽  
Sds Page ◽  
Ph Range ◽  
Digestive Fluid

Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-β-D-glucanase activity was confirmed in the supernatant of the insect’s digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications.

scholarly journals Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Si ◽  
Hongfei Ma ◽  
Yongjia Cao ◽  
Baokai Cui ◽  
Yucheng Dai
Keyword(s):  
Specific Activity ◽  
Endocrine Disrupting Chemicals ◽  
Environmental Pollutants ◽  
Fold Increase ◽  
Industrial Applications ◽  
White Rot ◽  
White Rot Fungus ◽  
Metallic Ions ◽  
Trametes Hirsuta ◽  
Ph Range

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

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 Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

2020 ◽  
Vol 25 (2) ◽  
pp. 127
Author(s):  
Kezia Abib Yerah Tjandra ◽  
Kartika Sari Dewi ◽  
Asrul Muhamad Fuad ◽  
Trisanti Anindyawati
Keyword(s):  
Pichia Pastoris ◽  
Trichoderma Reesei ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Endoglucanase Activity ◽  
Gap Promoter ◽  
Sds Page ◽  
Optimum Ph ◽  
High Concentrations

Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.

scholarly journals Exogenous Production of Cold-Active Cellulase From Psychrophilic Actinobacteria With Increased Cellulose Hydrolysis Efficiency

2021 ◽  
Author(s):  
Sivasankar Palaniappan ◽  
Poongodi Subramaniam ◽  
Sivakumar Kannan ◽  
Wahidah H Al-Qahtani ◽  
Arokiyaraj Selvaraj ◽  
...  
Keyword(s):  
Southern Ocean ◽  
Specific Activity ◽  
Terrestrial Ecosystems ◽  
Cellulose Hydrolysis ◽  
Industrial Applications ◽  
Polar Front ◽  
Biologically Active ◽  
Cellulolytic Activity ◽  
Cold Active ◽  
Cold Tolerant

Abstract Actinobacteria form the largest phylum consisting of diverse, ecologically unique and biologically active members. The actinobacteria are omnipresent and occur in various habitats such as cold environment, aquatic, desert and terrestrial ecosystems. Though the studies are available on actinobacteria at various habitats very few reports are available on cold tolerant/loving actinobacteria in the Southern Ocean part of the Antarctic Ocean. In this context, the present work was designed to isolate and characterize the actinobacteria in the Polar Front region of the Southern Ocean waters and species of Nocardiopsis and Streptomyces were identified. Among those, the psychrophilic actinobacterium, Nocardiopsis dassonvillei PSY13 was found to have good cellulolytic activity and it was further studied for the production and characterization of cold-active cellulase enzyme. The latter was found to have a specific activity of 6.36 U/mg and a molar mass of 48 kDa with a 22.9-fold purification and 5% recovery at an optimum pH of 7.5 and a temperature of 10 ºC. Given the importance of psychrophilic actinobacteria N. dassonvillei PSY13 can be further exploited for its benefits, meaning that the Southern Ocean harbours biotechnologically important microorganisms that can be further explored for versatile biotechnological and industrial applications.

scholarly journals Partial Purification of a Catalase from an Improved Nigerian Sorghum Grain Variety

2020 ◽  
pp. 30-37
Author(s):  
C. I. Nnamchi ◽  
B. C. Nwanguma ◽  
O. C. Amadi
Keyword(s):  
Crude Protein ◽  
Specific Activity ◽  
Partial Purification ◽  
Optimum Activity ◽  
Preliminary Purification ◽  
Sds Page ◽  
Cellular Detoxification ◽  
Activity Temperature ◽  
Ph Range ◽  
Sorghum Grain

Catalases are key components of cellular detoxification pathways that prevent the formation of highly reactive hydroxyl radicals through catalyzing the decomposition of hydrogen peroxide into water and molecular oxygen. Their presence in brewery grains prevent the inactivation of important brewery enzymes and also stop lipid peroxidation. To determine their occurrence and establish some of its properties in sorghum, which has become as an important brewery grain similar to barley, crude catalase was obtained from a sorghum grain variety. Preliminary purification of catalase from the sorghum grain variety used, NRL-3, showed that the enzyme was purified 3.2-fold from the crude protein to give a 49% yield of the partially purified enzyme, with a final specific activity of 32 Umg-1 proteins. There was also a positive indication of sorghum catalase presence on SDS PAGE with positive bands occurring between the range of 48-62 kDa. Therefore, the molecular weight of sorghum catalase most likely falls within the two bands. The enzyme showed a narrow pH range with optimum activity occurring at pH 7. Similarly, its optimum activity temperature occurred at 40°C.  This work is the first reported attempt at purifying catalase from sorghum.

scholarly journals Highly-Expressed Aspartic Protease from Aspergillus Fumigatus-Mediated Enzymolysis of Silkworm (Bombyx Mori) Pupae Protein

2021 ◽  
Author(s):  
Richard A. Herman ◽  
Chen Xie ◽  
Zi-Qian Zha ◽  
Zong-Nan Li ◽  
Jin-Zheng Wang ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Bombyx Mori ◽  
Specific Activity ◽  
Aspartic Protease ◽  
Degree Of Hydrolysis ◽  
Protease Gene ◽  
Silkworm Pupae ◽  
Sds Page ◽  
Ph Range ◽  
Hydrolysis Of

Abstract Aspartic protease emerges as an optimistic hydrolytic agent to obtain several protein hydrolysates. An aspartic protease gene from Aspergillus fumigatus Af293 was successfully expressed in Pichia pastoris (GS115) and its hydrolytic potentials on silkworm (Bombyx mori) pupae protein were determined. It was optimum at pH 4.0 and 50 °C and stable over pH range 4.0-5.0 and temperatures 45-55 °C with a specific activity of 8408.9 ± 305.6 U/mg. SDS-PAGE analysis revealed the molecular weight of the recombinant protease to be 45 kDa. The half-life (t1/2) of the recombinant protease at 40, 50, 60, and 70 °C was 30, 25, 35, and 20 min, respectively. The protease showed enhanced activity in the presence of Cu2+, Pb2+ and SDS. Its substrate specificity studies were revealed in the order of cleaving ability to Bovine Serum Albumin (BSA) > Silkworm pupae powder (SPP) > Casein > Casein sodium salt (CSS). Upon hydrolysis of silkworm pupae protein, it showed enhanced and plausible hydrolytic potentials, increasing the degree of hydrolysis to 50 ± 6.1% at 6 h, increased solubility by 80%, and improved functional properties. The stable characteristics and hydrolytic performance of the recombinant aspartic protease qualify it for industrial application, especially within the food and related industries.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

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

Marine Drugs ◽  
2020 ◽  
Vol 18 (4) ◽  
pp. 189 ◽  
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.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

scholarly journals Amylase from bacillus thuringiensis isolated from tapioca waste: isolation, partial purification and characterization

2016 ◽  
Vol 12 (1) ◽  
Author(s):  
Zusfahair Zusfahair ◽  
Dian Riana Ningsih ◽  
Dwi Kartika ◽  
Amin Fatoni
Keyword(s):  
Molecular Weight ◽  
Bacillus Thuringiensis ◽  
Specific Activity ◽  
Production Condition ◽  
Liquid Waste ◽  
Industrial Applications ◽  
Partial Purification ◽  
Production Time ◽  
Optimal Production ◽  
Sds Page

Microorganism enzymes are the most widely used in industrial applications. Tapioca liquid waste could be a great source of amylase-producing bacteria. The aim of this research was to isolate the amylase-producing bacteria form the tapioca waste, to produce amylase and to purify the resulted amylase. The screening, identification, and the optimal production condition of the amylase‒producing bacteria were studied.  The optimization of bacteria production growth phase and the amylase production time were investigated. The crude amylase was purified using ammonium sulfate fractionation followed by SDS PAGE electrophoresis to identify the molecular weight and to purity of the amylase. The amylase activity assay used was based on the measuring of resulted reducing sugar by Somogyi-Nelson method. The result showed that the amylase producing bacteria was identified as Bacillus thuringiensis. The exponential phase of the bacteria growth for bacteria adaptation before production was 18 h and the optimal production time of amylase enzyme was 24 h. The highest specific activity of the purified amylase was fraction (FHD) 40% with specific activity of 37.56 ± 0.38U/mg. The SDS PAGE of FHD 40% profile showed two clear bands with molecular weight of 32 kDa and 35 kDa respectively

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