scholarly journals Expression, Characterization and Structure Analysis of a New GH26 Endo-β-1, 4-Mannanase (Man26E) from Enterobacter aerogenes B19

2020 ◽  
Vol 10 (21) ◽  
pp. 7584
Author(s):  
Huijing Liu ◽  
Jie Liu ◽  
Tangbing Cui
Keyword(s):  
Enzyme Activity ◽  
Tertiary Structure ◽  
Enterobacter Aerogenes ◽  
Aromatic Rings ◽  
Sds Page ◽  
Catalytic Module ◽  
Wide Ph Range ◽  
Alkali Stability ◽  
Expression Characterization ◽  
Ph Range

β-mannanase is one of the key enzymes to hydrolyze hemicellulose. At present, most β-mannanases are not widely applied because of their low enzyme activity and unsuitable enzymatic properties. In this work, a new β-mannanase from Enterobacter aerogenes was studied, which laid the foundation for its further application. Additionally, we will further perform directed evolution of the enzyme to increase its activity, improve its temperature and pH properties to allow it more applications in industry. A new β-mannanase (Man26E) from Enterobacter aerogenes was successfully expressed in Escherichia coli. Man26E showed about 40 kDa on SDS-PAGE gel. The SWISS-MODEL program was used to model the tertiary structure of Man26E, which presented a core (α/β)8-barrel catalytic module. Based on the binding pattern of CjMan26 C, Man26E docking Gal1Man4 was investigated. The catalytic region consisted of a surface containing four solvent-exposed aromatic rings, many hydrophilic and charged residues. Man26E displayed the highest activity at pH 6.0 and 55 °C, and high acid and alkali stability in a wide pH range (pH 4–10) and thermostability from 40 to 50 °C. The enzyme showed the highest activity on locust bean gum, and the Km and Vmax were 7.16 mg mL−1 and 508 U mg−1, respectively. This is the second β-mannanase reported from Enterobacter aerogenes B19. The β-mannanase displayed high enzyme activity, a relatively high catalytic temperature and a broad range of catalytic pH values. The enzyme catalyzed both polysaccharides and manno-oligosaccharides.

scholarly journals Purification and Characterization of Strong Simultaneous Enzyme Production of Protease and α-Amylase from an Extremophile-Bacillus sp. FW2 and Its Possibility in Food Waste Degradation

Fermentation ◽  
2021 ◽  
Vol 8 (1) ◽  
pp. 12
Author(s):  
Van Hong Thi Pham ◽  
Jaisoo Kim ◽  
Jeahong Shim ◽  
Soonwoong Chang ◽  
Woojin Chung
Keyword(s):  
Food Waste ◽  
Environmental Conditions ◽  
Enzyme Production ◽  
Molecular Weights ◽  
Sds Page ◽  
Metabolic Products ◽  
Wide Ph Range ◽  
Waste Degradation ◽  
Ph Range

Microbial enzymes such as protease and amylase are valuable enzymes with various applications, widely investigated for their applications in degradation of organic waste, biofuel industries, agricultural, pharmaceuticals, chemistry, and biotechnology. In particular, extremophiles play an important role in biorefinery due to their novel metabolic products such as high value catalytic enzymes that are active even under harsh environmental conditions. Due to their potentials and very broad activities, this study isolated, investigated, and characterized the protease- and amylase-producing bacterial strain FW2 that was isolated from food waste. Strain FW2 belongs to the genus Bacillus and was found to be closest to Bacillus amyloliquefaciens DSM 7T with a similarity of 99.86%. This strain was able to degrade organic compounds at temperatures from −6 °C to 75 °C (but weak at 80 °C) under a wide pH range (4.5–12) and high-salinity conditions up to 35% NaCl. Maximum enzyme production was obtained at 1200 ± 23.4 U/mL for protease and 2400 ± 45.8 U/mL for amylase for 4 days at pH 7–7.5, 40–45 °C, and 0–10% NaCl. SDS-PAGE analysis showed that the molecular weights of purified protease were 28 kDa and 44 kDa, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively, and molecular weight of α-amylase was 55 kDa. Degradation food waste was determined after 15 days, observing a 69% of volume decrease. A potential commercial extremozyme-producing bacteria such as strain FW2 may be a promising contributor to waste degradation under extreme environmental conditions.

Purification and properties of a nonspecific β-fructofuranosidase (inulinase) from the mushroom Panaeolus papillonaceus

1987 ◽  
Vol 33 (6) ◽  
pp. 520-524 ◽  
Author(s):  
Khana Mukherjee ◽  
S. Sengupta
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Activity Ratio ◽  
Optimum Temperature ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Inulinase Activity ◽  
Wide Ph Range ◽  
Total Molecular Weight ◽  
Ph Range

A nonspecific β-fructofuranosidase (inulinase) was purified to electrophoretic homogeneity from the culture filtrate of the mushroom Panaeolus papillonaceus. The enzyme is the first purified from a basidiomycete and consists of two subunits with a total molecular weight of 116 000. It is most active on sucrose, then on raffinose, stachyose, and inulin, in decreasing order. The sucrase/inulinase activity ratio (S/I) is 5.7. Fructose was detected as the liberated sugar from raffinose, stachyose, and inulin. The enzyme is highly thermostable with an optimum temperature range of 60–65 °C and a pH optimum of 6.0. The enzyme is stable over the pH range 4–10, and is also active over a wide pH range, exhibiting 50% activity even at pH 8.5. Iodoacetate, azide, and EDTA, at 20 mM concentration, and 1% (w/v) SDS have no effect on enzyme activity, whereas Ag+ and Hg2+ at 2 mM are highly inhibitory.

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.

Thermostable alkaline protease from newly isolated Vibrio sp.: extraction, purification and characterisation

Biologia ◽  
2012 ◽  
Vol 67 (4) ◽  
Author(s):  
Ponnambalam Subhashini ◽  
Neelamegam Annamalai ◽  
Ayyappan Saravanakumar ◽  
Thangavel Balasubramanian
Keyword(s):  
Enzyme Activity ◽  
Organic Solvents ◽  
Alkaline Protease ◽  
Mangrove Sediments ◽  
Sds Page ◽  
Vellar Estuary ◽  
Commercial Detergents ◽  
The Stability ◽  
Ph Range ◽  
Triton X

AbstractAn extracellular alkaline protease-producing Vibrio sp. was isolated from mangrove sediments of Vellar estuary. A 9.36-fold purification was achieved by a three-step purification procedure and the molecular weight of the enzyme was determined as 33 kDa by SDS-PAGE. The enzyme was active in a broad range of pH (6.0–11.0) and temperature (30–70°C), the optimum being at pH 9.0 and temperature 55°C. The enzyme was stable at alkaline pH range of 9–11 and up to a temperature of 60°C, after incubation for 1 h. Metals like Co2+, Hg2+, Ni2+ and Cu2+ inhibited the enzyme activity, whereas Fe2+, Ca2+ and Mn2+ were found to enhance the activity. The protease was found to be highly stable in the presence of oxidizing agents like H2O2, detergents such as SDS and Triton-X-100 and also some of the commonly used commercial detergents. The organic solvents like xylene, isopropanol, hexane and benzene were found to enhance as well as stabilize the enzyme activity. The extracellular production of the enzyme, the pH and thermal stability, and the stability in presence of oxidants, surfactants, commercial detergents and organic solvents, altogether suggest that it can be used as a laundry additive.

Polarographic and spectrophotometric behaviour of some N-p-phenyl substituted benzamidines

1991 ◽  
Vol 56 (12) ◽  
pp. 2791-2799 ◽  
Author(s):  
Juan A. Squella ◽  
Luis J. Nuñez-Vergara ◽  
Hernan Rodríguez ◽  
Amelia Márquez ◽  
Jose M. Rodríguez-Mellado ◽  
...  
Keyword(s):  
Spectrophotometric Study ◽  
Absorption Bands ◽  
Electrochemical Parameters ◽  
Wide Ph Range ◽  
Group A ◽  
Substituent Constants ◽  
Ph Range ◽  
The Waves

Five N-p-phenyl substituted benzamidines were studied by DC and DP polarography in a wide pH range. Coulometric results show that the overall processes are four-electron reductions. Logarithmic analysis of the waves indicate that the process are irreversible. The influence of the pH on the polarographic parameters was also studied. A UV spectrophotometric study was performed in the pH range 2-13. In basic media some variations in the absorption bands were observed due to the dissociation of the amidine group. A determination of the pK values was made by deconvolution of the spectra. Correlations of both the electrochemical parameters and spectrophotometric pK values with the Hammett substituent constants were obtained.

High degradation efficiency of sulfamethazine with the dual-reaction-center Fe-Mn-SiO2 Fenton-like nanocatalyst in a wide pH range

2021 ◽  
Author(s):  
Manoj Kumar Panjwani ◽  
Qing Wang ◽  
Yueming Ma ◽  
Yuxuan Lin ◽  
Feng Xiao ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Reaction Center ◽  
Degradation Efficiency ◽  
Heterogeneous Fenton ◽  
Wide Ph Range ◽  
Ph Range

The development of a heterogeneous Fenton-like catalyst, possessing high degradation efficiency in a wide pH range, is crucial for wastewater treatment. The Fe-Mn-SiO2 catalyst was designed, and prepared by a...

scholarly journals Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Preeti Anand ◽  
Jay Prakash Pandey ◽  
Dev Mani Pandey
Keyword(s):  
San Francisco ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Evolutionary Genetics ◽  
Imaging Analysis ◽  
Silk Cocoon ◽  
Natural Color ◽  
Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

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