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 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 thermotolerant lipase with broad pH profiles isolated from an AntarcticPseudomonassp strain AMS3

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2420 ◽  
Author(s):  
Wahhida Latip ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Adam Thean Chor Leow ◽  
Fairolniza Mohd Shariff ◽  
Mohd Shukuri Mohamad Ali
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Organic Solvent ◽  
Divalent Metal ◽  
Single Step ◽  
Divalent Metal Ions ◽  
Gene Encoding ◽  
Affinity Tags ◽  
Ph Range

A gene encoding a thermotolerant lipase with broad pH was isolated from an AntarcticPseudomonasstrain AMS3. The recombinant lipase AMS3 was purified by single-step purification using affinity chromatography, yielding a purification fold of approximately 1.52 and a recovery of 50%. The molecular weight was approximately ∼60 kDa including the strep and affinity tags. Interestingly, the purified Antarctic AMS3 lipase exhibited broad temperature profile from 10–70 °C and stable over a broad pH range from 5.0 to pH 10.0. Various mono and divalent metal ions increased the activity of the AMS3 lipase, but Ni2+decreased its activity. The purified lipase exhibited the highest activity in the presence of sunflower oil. In addition, the enzyme activity in 25% v/v solvents at 50 °C particularly to n-hexane, DMSO and methanol could be useful for catalysis reaction in organic solvent and at broad temperature.

scholarly journals Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

2003 ◽  
Vol 69 (2) ◽  
pp. 980-986 ◽  
Author(s):  
Dae Heoun Baek ◽  
Seok-Joon Kwon ◽  
Seung-Pyo Hong ◽  
Mi-Sun Kwak ◽  
Mi-Hwa Lee ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Optimum Temperature ◽  
Gene Encoding ◽  
Ph Range

ABSTRACT A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The k cat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+.

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

2016 ◽  
Vol 88 (suppl 1) ◽  
pp. 479-487 ◽  
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.

scholarly journals Characterization of cellulase from E. coli BPPTCC-EGRK2

2018 ◽  
Vol 52 ◽  
pp. 00024
Author(s):  
Mas Gunawan Haryanto ◽  
Siswa Setyahadi ◽  
Muhammad Sahlan ◽  
Masafumi Yohda ◽  
Yosuke Fukutani ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Animal Feed ◽  
Maximum Velocity ◽  
Cellulase Production ◽  
Luria Bertani ◽  
E Coli ◽  
Sds Page ◽  
Weight Analysis

Cellulase enzymes are widely used in various industries such as detergent industry, bioethanol, animal feed, textile and paper. This research focused on characterization of cellulase produced from Eschericia coli BPPT-CC EgRK2, which is a recombinant that can produce protein enzymes endo- β-1,4-glucanase. Eschericia coli BPPT-CC EgRK2 was cultured in 1 litre liquid medium Luria Bertani. Because the bacteria is intracellular, sonication is needed for cell disruption to get the cellulase enzyme. The enzyme activity was then analyzed by CMC substrate at different concentration. The protein content analysis was carried out using Bradford method; the molecular weight analysis was done using SDS-PAGE; while the enzyme kinetics was plotted using Michaelis-Menten model. Our results showed the highest enzyme activity was 2.403 U/ml and the protein concentration was 5.352 mg/ml. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for CMC substrate hydrolysis were 0.07 μmol/ml and 2.49 μmol/ml/sec, respectively. The cellulase molecular weight was 58 kDa using SDS-PAGE with 7.5% stacking gel. The results indicated that Eschericia coli BPPT-CC EgRK2 is a promising renewable source for cellulase production for industrial application.

Purification and Characterization of a Bioscouring Pectate Lyase from Paenibacillus sp. WZ008 with High Activity on Pectin

2012 ◽  
Vol 441 ◽  
pp. 457-461
Author(s):  
Xiang Xian Ying ◽  
Li Na Chen ◽  
Mei Lan Yu ◽  
Qun Xue ◽  
Zhao Wang
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
High Activity ◽  
Culture Supernatant ◽  
Pectate Lyase ◽  
Maximal Activity ◽  
Polygalacturonic Acid ◽  
Purification And Characterization ◽  
Optimum Activity

An extracellular pectate lyase was purified from the culture supernatant of Paenibacillus sp. WZ008 grown in the pectin-containing medium. The enzyme was purified to homogeneity in three steps and found to have a molecular weight of around 45 kDa. Highly methylated pectin was the optimum substrate in the case of no Ca2+ addition while the enzyme exhibited the maximal activity on polygalacturonic acid in the presence of 4 mM Ca2+. The purified enzyme demonstrated the optimum activity at a temperature range of 55-60°C and pH 9.6. The Ca2+ ion enhanced the enzyme activity but Mn2+, Ba2+ and EDTA strongly inhibited it.

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.

scholarly journals Characterization of a novel laccase purified from the fungus Hohenbuehelia serotina and its decolourisation of dyes.

2015 ◽  
Vol 63 (2) ◽  
Author(s):  
Yingyin Xu ◽  
Yuxiao Lu ◽  
Rui Zhang ◽  
Hexiang Wang ◽  
Qinghong Liu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Inhibitory Effect ◽  
Single Band ◽  
White Rot ◽  
White Rot Fungus ◽  
Sds Page

A novel laccase was purified from the white rot fungus, Hohenbuehelia serotina, to investigate the applications of this laccase in the decoloration of various dyes. SDS-PAGE revealed a single band of this laccase corresponding to a molecular weight of approximately 57.8 kDa. The enzyme showed activity towards several substrates, the most sensitive of which was 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). The highest enzymatic activity using ABTS as a substrate was observed at pH 6.8 and 30°C. The enzyme activity was found to be significantly enhanced in the presence of Zn(2+) ions and inhibited by Fe(2+) ions. Moreover, SDS and β-mercaptoethanol were inhibitory, and inhibition by L-cysteine was observed while EDTA and DMSO had almost no inhibitory effect. The laccase could effectively decolorize seven different dyes within 30 minutes at 40°C.

scholarly journals Purification, Characterization and De-Staining Potentials of a Thermotolerant Protease Produced by Fusarium oxysporum

2019 ◽  
Vol 64 (4) ◽  
pp. 539-547
Author(s):  
Mohammed Inuwa Ja’afaru ◽  
Konjerimam Ishaku Chimbekujwo ◽  
Obinna Markraphael Ajunwa
Keyword(s):  
Enzyme Activity ◽  
Fusarium Oxysporum ◽  
Treatment Time ◽  
Specific Activity ◽  
Total Yield ◽  
Tween 20 ◽  
Optimum Temperature ◽  
Industrial Enzymes ◽  
Sds Page ◽  
Optimum Ph

Proteases are important industrial enzymes and fungi prove to be good sources of such enzymes. Purification techniques are however necessary for increased specificity in activity and better industrial value. Based on this, a protease produced by a Fusarium oxysporum was purified to homogeneity by Sephadex G-200 column and α–casein agarose chromatography. The enzyme had a molecular weight of 70 kDa in SDS-PAGE. Purified Fusarium oxysporum protease had a specific activity of 93.88 U/mg protein. The purification magnitude was 7.7 and the total yield was 20 %. Purified protease had an optimum pH of 5.0 while the optimum temperature was 40 °C. The enzyme was also thermotolerant (approximately 100 % at 40 °C for 2 h). The enzyme activity was stimulated by surfactants and metal ions like, Tween-20 and Mg2+. Enzyme activity was inhibited in presence of PMSF and EDTA. Casein was found to be the best substrate for protease activity of Fusarium oxysporum FWT1. Protease were tested upon blood stain for de-clotting of blood and was found to exhibit good de-clotting and de-staining activity after 15 minutes treatment time.

Enzymological Characterization of Secreted Proteinases from Candida parapsilosis and Candida lusitaniae

2001 ◽  
Vol 66 (11) ◽  
pp. 1707-1719 ◽  
Author(s):  
Olga Hrušková-Heidingsfeldová ◽  
Jiří Dostál ◽  
Petr Hamal ◽  
Jarmila Pazlarová ◽  
Tomáš Ruml ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Candida Parapsilosis ◽  
Yeast Culture ◽  
Opportunistic Pathogens ◽  
Novel Drugs ◽  
Sds Page ◽  
Candida Lusitaniae ◽  
Culture Supernatants

Opportunistic pathogens of the genusCandidaproduce secreted aspartic proteinases (Saps) that are considered as one of the virulence factors. While Saps ofC. albicansandC. tropicalishave been studied extensively, information concerning proteinases secreted by other pathogenicCandidaspecies is scarce. To our knowledge, enzymologic characterizations of Sap ofC. parapsilosis(Sapp1p) andC. lusitaniae(Saplp) are limited. We have purified Saps ofC. parapsilosisandC. lusitaniaefrom yeast culture supernatants and detected only one gene product of both Sapp1p and Saplp using isoelectric focusing and N-terminal sequencing. Molecular weight of Saplp determined by SDS-PAGE is approximately 42 000. For the highest enzyme activity, both Sapp1p and Saplp require pH ranging from 3 to 4 and temperature between 27-40 °C for Saplp and 45 °C for Sapp1p, respectively. At physiological temperature, both the proteinases are stable. The characterization of Saps of clinical isolates ofC. parapsilosisandC. lusitaniaeshould contribute to design of novel drugs specifically targeted on proteinases.

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