Screening and Characterization of a Mutant Fungal Aspartic Proteinase from Mucor pusillus

In this study, site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet (MPR) in order to find a potential substitution of commercial chymosin. Mutant G186D/E13D screened from thousands of mutants showed a significant milk-clotting activity (MCA). Mutant G186D/E13D rennet was purified and characterized. The molecular weight was estimated to be 44 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pH (5.0-7.0) and 60ºC. The enzyme was inhibited by metal ions (Fe2+, Fe3+, Cu+ and Zn2+), 1.10-Phenantrolin and pepstatin A. Further texture analysis of types of cheddar cheese made by non-mutant rennet, mutant (G186D/E13D) rennet and commercial rennet suggested that the soluble nitrogen content and hardness of cheddar cheese made by chimeric mutant rennet was decreased without any significant change in flavor between these cheeses. The result implicated that, to some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably as a candidate for a useful milk coagulant.

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

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.252-256.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 252-256 ◽

Cited By ~ 19

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 ).

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Characterization of Gelatin Profile of Chicken Broiler (Gallus domestica) Bone Using SDS-PAGE Electrophoresis

ALCHEMY ◽

10.18860/al.v7i1.7437 ◽

2019 ◽

Vol 7 (1) ◽

pp. 7 ◽

Cited By ~ 2

Author(s):

Dewi Yuliani ◽

Dhienda Risa Awalsasi ◽

Akyunul Jannah

Keyword(s):

Ammonium Sulfate ◽

Protein Concentration ◽

Peptide Fragments ◽

Strong Base ◽

Chicken Bone ◽

Sds Page ◽

Hydrolysis Of ◽

Β Chain ◽

Page Electrophoresis

Gelatin, a proteinaceous additive, is obtained from hydrolysis of collagen in the bone, hide and skin of animals. As natural product, gelatin has been applied in many industries with various functions. This study attempt to characterize gelatin profile of broiler chicken (Gallus domestica) using SDS-PAGE electrophoresis. The chicken bone was pretreated using a strong base, sodium hydroxide, producing type B gelatin. The gelatin was purified through precipitation using the variation of ammonium sulfate concentrations (40-70%) and dialysis using cellophane membrane. The purified gelatin was characterized through SDS-PAGE electrophoresis. Based on electrophoresis visualization, reduction of band intensity by ammonium sulfate 40% showed removal of small peptide fragments. The remained gelatin showed two major bands, α-chains and a β-chain with the respective molecular weight of ~135 and ~245 kDa. The protein content of the unpurified gelatin (E1) was 71.65±0.60 mg/L. The purified E1 gelatins by 40-70% of ammonium sulfate addition contained 61.42±3.90, 60.45±1.36, 59.89±0.24, and 55.32±1.05 mg/L of protein concentration, respectively. Keywords: chicken bone, gelatin profile, protein electrophoresis

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Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

Biochemical Journal ◽

10.1042/bj3160841 ◽

1996 ◽

Vol 316 (3) ◽

pp. 841-846 ◽

Cited By ~ 29

Author(s):

Stuart M. PITSON ◽

Robert J. SEVIOUR ◽

Barbara M. McDOUGALL ◽

Bruce A. STONE ◽

Maruse SADEK

Keyword(s):

Molecular Mass ◽

Filamentous Fungus ◽

Gel Filtration ◽

Purification And Characterization ◽

Eisenia Bicyclis ◽

Ph Optimum ◽

Hydrolysis Products ◽

Sds Page ◽

Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

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Purification and characterization of a new endoglucanase from Clostridium thermocellum

Biochemical Journal ◽

10.1042/bj2830069 ◽

1992 ◽

Vol 283 (1) ◽

pp. 69-73 ◽

Cited By ~ 17

Author(s):

M P M Romaniec ◽

U Fauth ◽

T Kobayashi ◽

N S Huskisson ◽

P J Barker ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Reducing Sugars ◽

Rapid Decrease ◽

Purification And Characterization ◽

Temperature Optimum ◽

Apparent Homogeneity ◽

Sds Page ◽

Hydrolysis Of ◽

Optimal Ph

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.

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Characterization of Thermostable Amylase From Halophilic Lactobacillus Plantarum TS1

10.21203/rs.3.rs-565599/v1 ◽

2021 ◽

Author(s):

Sania Riaz ◽

Anum Fareed ◽

Habiba Zaffar ◽

Shafique Ur Rehman ◽

Muhammad Jamil ◽

...

Keyword(s):

Lactobacillus Plantarum ◽

Saline Soil ◽

Salt Range ◽

Sds Page ◽

Wide Range ◽

Two Temperatures ◽

Harsh Conditions ◽

Thermostable Amylase ◽

Important Enzyme

Abstract Amylase is an important enzyme use extensively in various industrial processes. It is mainly involved in the catalysis of starch that requires harsh conditions; therefore, it is required to isolate amylases with unique properties that makes it more applicable. Extremophiles are the major resource of such enzymes; therefore, amylase positive strains were isolated from the saline soil where the temperature is also exceptionally high. Five amylase positive strains were isolated from the Karak salt range, Kohat Pakistan and were identified by phylogenetic analysis. DNS based assay was employed to compare the activities of different amylases obtained from five strains while using the starch as a substrate. The amylase obtained from Lactobacillus plantarum TS1 was found to be more efficient, which was purified and characterized. The SDS-PAGE of purified amylase showed a single band with an estimated size of 10 kDa. The kinetic parameters were measured at two temperatures i.e. at 37 °C and 50 °C. The Kcat as well as the Kcat/KM were found to be high when temperature increased from 37 °C to 50 °C. Amylase was active at wide range of temperature as well as pH and work efficiently in the presence of salts and various organic solvents.

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PRODUCTION AND PARTIAL CHARACTERIZATION OF A MILK-CLOTTING PROTEINASE PRODUCED BY BACILLUS SUBTILIS SMDFS-2B MN715837 IN SUBMERGED CULTURES

Bacterial Empire ◽

10.36547/be.257 ◽

2021 ◽

Vol 4 (2) ◽

pp. e257

Author(s):

Jermen Mamo ◽

Martin Kangwa ◽

Hector-Marcelo Fernandez-Lahore ◽

Fassil Assefa

Keyword(s):

Bacillus Subtilis ◽

Crude Enzyme ◽

Partial Characterization ◽

Effect Of Temperature ◽

Submerged Cultures ◽

Milk Clotting ◽

Milk Clotting Protease ◽

Pepstatin A ◽

Milk Clotting Activity

This study focused on the production and partial characterization of a milk-clotting protease produced by Bacillus subtilis SMDFS 2B in submerged cultures, under partially optimized conditions. The crude enzyme was recovered in the culture supernatant and concentrate was produced after cell removal and subsequent dialysis. Inhibition studies were conducted employing four distinct protease inhibitors: Pepstatin-A, Phenylmethane-sulphonyl-fluoride (PMSF), Ethylenediaminetetraacetic acid (EDTA), and iodoacetamide (IA). The effect of temperature, pH, metal ions and substrate concentration on milk-clotting activity were also evaluated. The thermal stability of the enzyme was determined by incubating the crude enzyme at a temperature value ranging from 35 oC to 60 oC. Similarly, pH stability was determined at pH values ranging between 4.5 and 8.0. The highest milk-clotting activity was observed at a temperature of 55 oC and pH 5.5. The crude enzyme preparation remained stable on incubation at 35 oC and 40 oC for 15 min and at pH 5.5. The enzyme also showed the lowest residual milk-clotting activity in the presence of EDTA (7.94%) and Pepstatin-A (26.71%). The addition of Mg2+ and Mn2+ significantly increased milk-clotting activity. The enzyme also showed an elevation in its apparent milk-clotting activity upon increasing the substrate (skim-milk) concentration. Thus, the milk-clotting protease produced by B. subtilis SMDFS 2B by submerged fermentation revealed some interesting milk-clotting characteristics. This may open the way for applications in the food and dairy industries.

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Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

Jurnal Agritech ◽

10.22146/agritech.17004 ◽

2017 ◽

Vol 37 (1) ◽

pp. 31

Author(s):

Fitria Fitria ◽

Nanik Rahmani ◽

Sri Pujiyanto ◽

Budi Raharjo ◽

Yopi Yopi

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Centrifugal Filter ◽

Sds Page ◽

Enzyme Specific Activity ◽

Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the xylooligosaccharide types from xylan hydrolysis by this enzyme. Based on this research, the optimum time for enzyme production occurred at 96 hours with the enzyme activity of 6.275 U/mL and enzyme specific activity of 5.093 U/mg. The specific activities were obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0 U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram showed that the molecular weight of xylanase protein were about 25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji hidrolisis untuk mengetahui jenis xilooligosakarida yang dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96 dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil pemekatan dengan amicon® ultra-15 centrifugal filter devices, kromatografi filtrasi gel dan kromatografi penukar anion mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

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Recombinant expression and characterization of Solanum tuberosum aspartic protease

SURG Journal ◽

10.21083/surg.v3i1.1032 ◽

2009 ◽

Vol 3 (1) ◽

pp. 19-25

Author(s):

Lauren Agro ◽

Brian Bryska ◽

Rickey Yada

Keyword(s):

Solanum Tuberosum ◽

Disulfide Bonds ◽

Inclusion Bodies ◽

Signal Sequence ◽

Recombinant Expression ◽

Aspartic Proteinase ◽

Wild Type ◽

Sds Page ◽

Hemoglobin Degradation

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM

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Characterization of the Family I inorganic pyrophosphatase fromPyrococcus horikoshiiOT3

Archaea ◽

10.1155/2005/591628 ◽

2005 ◽

Vol 1 (6) ◽

pp. 385-389 ◽

Cited By ~ 10

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.

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THE CHARACTERIZATION OF LACTOBACILLI FROM CHEDDAR CHEESE: I. AN EVALUATION OF PHYSIOLOGICAL AND BIOCHEMICAL TESTS

Canadian Journal of Microbiology ◽

10.1139/m62-094 ◽

1962 ◽

Vol 8 (5) ◽

pp. 727-735 ◽

Cited By ~ 6

Author(s):

R. E. Smith ◽

J. D. Cunningham

Keyword(s):

Preliminary Investigation ◽

Skim Milk ◽

Cheddar Cheese ◽

Titratable Acidity ◽

Lactobacillus Brevis ◽

Lactobacillus Helveticus ◽

Biochemical Tests ◽

Morphological Studies ◽

Hydrolysis Of

Characterization studies were conducted on 230 cultures of lactobacilli isolated from Canadian Cheddar cheese, and on an additional 15 named cultures from various sources. Preliminary investigation included reactions with 19 carbohydrates, yeast glucose litmus milk, and arginine, hippurate, and aesculin broths. This resulted in the appearance of six major groups, tentatively designated as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus fermenti, and an unclassifiable group. Subgroups of the divisions were noted. Sixty-eight cultures were chosen for detailed study. Tests performed included the production of catalase, nitrite, hydrogen sulphide, indole, and polysaccharide; the hydrolysis of starch, gelatin, Tweens 40 and 60, polypectate, and casein; and tolerance of growth temperatures, sodium chloride, and phenol. Titratable acidity in skim milk was determined, and morphological studies were carried out. Accumulated data indicated that the group previously designated as L. helveticus, and the unclassified group, consisted of variants of L. plantarum, L. casei, or intermediates.

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