Characterization of Genetically Engineered Linamarase (β-glucosidase) from Saccharomyces cerevisiae

The characterization parameters of genetically engineered linamarase (β-glucosidase) from Saccharomyces cerevisiae due to action of the enzyme on linamarin as influenced by degree of purification, pH and temperature were investigated. Commercial native linamarase (CNLIN) was used as control. Linamarase genes (chromosomal DNA) and plasmids (circular DNA) isolated from bitter cassava and yeast respectively were restricted and ligated to produce recombinant genes (r-DNA). The r-DNA were introduced into the nucleus of CaCl2 induced competent Saccharomyces cerevisiae cells which transformed into strains capable of producing genetically engineered linamarase (GELIN). Recombinant S. cerevisiae cells at the stationary phase of growth were recovered, homogenized and centrifuged to obtain crude extracts designated as GELIN0. Carboxy methyl cellulose, diethyl amino-ethyl-sephadex and diethyl amino-ethyl-cellulose were used to purify the crude extracts resulting in GELIN1, GELIN2 and GELIN3, respectively. The physical characterization parameters of the enzyme extracts such as impurity levels, molecular weights (Mwt), number of isoenzyme, sulphur amino acids (methionine and cysteine) and the electrical charges were evaluated using standard methods. The ability of the enzyme extracts and a commercial native linamarase (CNLIN) to hydrolyse cyanogenic glucosides was challenged using linamarin (cassava) as substrates for characterization of activity kinetic profiles such as optimum pH (pHopt), temperature (Topt), total activity, specific activity, purity fold, yield and efficiency ratio. The results indicated that the genetically engineered linamarase(β-glucosidase) consisted of 3 isoenzyme forms. Purification conferred different ionic charges of zero to GELIN0, unit positive charge GELIN1, and unit negative charge to GELIN2 and GELIN3 respectively. Ranges for other parameters were Mwt (22,000-26,000 Daltons), insoluble protein impurity (0.4 -3.5 mg/100g sample) and purity fold (11.5 -1.0) for GELIN3 - GELIN0). Methionine and cystiene varied from 2.0 to 2.6% and 3.0 to 20% respectively (CNLIN - GELIN3). The native commercial enzyme (CNLIN) acted only at pH 6.8 on linamarin with pHopt and Topt of 6.8 and 35 oC respectively. The wide pH tolerance and specific activity towards linamarin degradation suggest a possible use of the genetically engineered linamarase from S. cerevisiae in detoxification of cassava for increased production exportation of cassava-based food products.

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Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

Biochemical Journal ◽

10.1042/bj1910117 ◽

1980 ◽

Vol 191 (1) ◽

pp. 117-124 ◽

Cited By ~ 14

Author(s):

R Zecher ◽

H U Wolf

Keyword(s):

Specific Activity ◽

Total Activity ◽

Partial Purification ◽

Half Maximum ◽

Purification And Characterization ◽

Dimeric Molecule ◽

Maximum Inhibition ◽

Optimum Ph ◽

Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

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Angelicin as the Principal Allelochemical in Heracleum sosnowskyi Fruit

Natural Product Communications ◽

10.1177/1934578x1501000517 ◽

2015 ◽

Vol 10 (5) ◽

pp. 1934578X1501000 ◽

Cited By ~ 3

Author(s):

Maryia Mishyna ◽

Nikolai Laman ◽

Valery Prokhorov ◽

Yoshiharu Fujii

Keyword(s):

Inhibitory Activity ◽

Specific Activity ◽

Lolium Multiflorum ◽

Distribution Patterns ◽

Total Activity ◽

Cold Stratification ◽

Hypocotyl Growth ◽

Heracleum Sosnowskyi ◽

Crude Extracts ◽

Inhibitory Potential

Distribution patterns of furocoumarins in fruits of the invasive species Heracleum sosnowskyi Manden. (Sosnowskyi's hogweed) during a cold stratification period were investigated. Angelicin, bergapten, methoxalen and imperatorin were mainly localized in the fruit coats and their content varied depending on the fruit source. Cold stratification treatment (90 days, 2–3°C) reduced the content of furocoumarins in the fruit coats by more than two times, compared with those before stratification. The specific activity of the detected furocoumarins and total activity of crude extracts were evaluated using Lactuca sativa, as acceptor plant. Crude extracts obtained from fruit coats and seeds of H. sosnowskyi suppressed 50% of radicle and hypocotyl growth of lettuce seedlings at the concentration range of 1.0–1.7 mg/mL. The inhibitory activity of angelicin was proved to be the highest compared with the other tested furocoumarins, and the inhibitory activity of crude extracts could be explained mainly by the presence of angelicin. Both, monocots ( Lolium multiflorum, Phleum pratensis, Festuca pratesis, Lolium perenne) and dicots ( Tripholium repens, Trifolium pretense) were found to be sensitive to the exudates of whole H. sosnowskyi fruits. Thus, we assume, that high inhibitory potential of furocoumarins, especially angelicin, at high seed productivity of H. sosnowskyi might have an ecological significance in plant-plant interaction.

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Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661517 ◽

1986 ◽

Vol 55 (02) ◽

pp. 178-183

Author(s):

N Shimada ◽

M Tsubokura ◽

N Kimura

Keyword(s):

Molecular Weight ◽

Adenylate Cyclase ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

High Specific Activity ◽

Total Activity ◽

Human Platelets ◽

Protein Profiles

SummaryIsolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.

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Molecular genetic properties of the yeast Torulaspora pretoriensis: Characterization of chromosomal DNA and genetic transformation by Saccharomyces cerevisiae-based plasmids

Current Genetics ◽

10.1007/bf00313426 ◽

1995 ◽

Vol 27 (2) ◽

pp. 131-134 ◽

Cited By ~ 8

Author(s):

Yuji Oda ◽

Kenzo Tonomura

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Transformation ◽

Molecular Genetic ◽

Chromosomal Dna

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Characterization of partially purified phospholipase C from human platelet membranes

Biochemical Journal ◽

10.1042/bj2480095 ◽

1987 ◽

Vol 248 (1) ◽

pp. 95-101 ◽

Cited By ~ 18

Author(s):

Y Banno ◽

Y Nozawa

Keyword(s):

Phospholipase C ◽

Membrane Fraction ◽

Specific Activity ◽

Human Platelet ◽

Gel Filtration ◽

Total Activity ◽

Hydrolytic Activity ◽

Platelet Membrane ◽

Dependent Manner

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.

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Studies on the Characterization of Esterase from Different Pisum sativum L. (Fabaceae) Varieties

Revista de Chimie ◽

10.37358/rc.20.9.8316 ◽

2020 ◽

Vol 71 (9) ◽

pp. 47-54

Author(s):

Anushree Srinivasa Murthy ◽

Ramya Manjunath ◽

Tulasi Dhondale Prakash ◽

Mahesh Krishna Reddy ◽

Krishna Rao Jagarlamudi ◽

...

Keyword(s):

Pisum Sativum ◽

Specific Activity ◽

Total Activity ◽

Stored Products ◽

Optimum Temperature ◽

Kinetic Characteristics ◽

Qualitative And Quantitative ◽

Dimethyl Phosphate ◽

Pisum Sativum L

Pisum sativum L. (Fabaceae) is important in diet due to its fiber content, protein, starch, trace elements and phytochemical substances. Dichlorvos (2, 3-dichlorovinyl dimethyl phosphate) is commonly known as organophosphates belong to the classes of insecticides that are used to control households and stored products insects. The aim of this study was to analyze the kinetic characteristics of esterase enzyme from four different varieties of Pisum sativum L. using qualitative and quantitative method and also the docking position of esterase with Dichlorvos by insilico method. According to our study the total activity was highest for Arka Nirmala and Arka Priya varieties. The specific activity was highest for Arka Mayur variety. Km and Vmax, were found to be higher for Arka Uttam. The optimum temperature and pH for all the varieties were found to be same, 40�C and 7.5 respectively. The inhibitor studies showed Arka Nirmala was more sensitive to inhibitor. From the fitness and interaction profile it was found that the inhibitor dichlorovos is an effective pesticide which can be used to inhibit esterase activity. It has one hydrogen bond of H-S and had an amino acid residue at ARG57.

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Application of dhurrin for kinetics and thermodynamic characterization of linamarase (β-glucosidase) genetically engineered from Saccharomyces cerevisiae.

Agro-Science ◽

10.4314/as.v13i3.7 ◽

2015 ◽

Vol 13 (3) ◽

pp. 49

Author(s):

JK Ikya ◽

CC Arishu ◽

JOI Ayatse

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetically Engineered ◽

Thermodynamic Characterization ◽

Kinetics And Thermodynamic

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Involvement of Carnosic Acid in the Phytotoxicity of Rosmarinus officinalis Leaves

Toxins ◽

10.3390/toxins10120498 ◽

2018 ◽

Vol 10 (12) ◽

pp. 498 ◽

Cited By ~ 5

Author(s):

Kwame Appiah ◽

Hossein Mardani ◽

Richard Omari ◽

Vincent Eziah ◽

John Ofosu-Anim ◽

...

Keyword(s):

Specific Activity ◽

Control Measure ◽

High Specific Activity ◽

Total Activity ◽

Inhibitory Effect ◽

Rosmarinus Officinalis ◽

Carnosic Acid ◽

High Concentration ◽

Crude Extracts ◽

Test Plants

Weeds are rapidly developing resistance to synthetic herbicides, and this can pose a threat to the ecosystem. Exploring allelopathic species as an alternative weed control measure can help minimize the ecological threat posed by herbicide-resistant weeds. In this study, we aimed to evaluate the contribution of some polyphenols to the allelopathy of rosemary (Rosmarinus officinalis L.). The phytotoxic effects of rosemary (leaves, roots, inflorescences, and stems) crude extracts were tested on lettuce (Lactuca sativa L.). Soils incorporated with dried rosemary leaves were also tested on test plants. Reversed-phase high-performance liquid chromatography (HPLC) analysis was used to determine the content of some polyphenols (caffeic, ferulic, gallic, rosmarinic, carnosic, and chlorogenic acids) in rosemary. The specific activity and total activity of crude extracts and individual compounds were evaluated using lettuce. The crude extract of rosemary leaves showed the highest growth inhibitory effect among the rosemary organs tested. Soil amended with rosemary leaf debris reduced the dry matter and seed emergence of lettuce. Carnosic acid was the main compound detected in rosemary leaves and had a high specific activity when tested on lettuce. During the seed germination period, there was observed filter paper coloration among the test plants treated with carnosic acid (250 μg/mL). The high concentration and strong inhibitory effect of carnosic acid could explain the inhibitory activity of the rosemary leaf extract. Hence, we conclude based on the total activity estimation that carnosic acid among the other tested compounds is the major allelochemical in rosemary leaves.

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Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae

Journal of Bacteriology ◽

10.1128/jb.152.1.323-331.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 323-331

Author(s):

L Naumovski ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Cloning ◽

Dna Isolation ◽

Excision Repair ◽

Restriction Analysis ◽

Uv Resistance ◽

Chromosomal Dna ◽

Uv Sensitivity ◽

Resultant Plasmid

We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size.

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Purifikasi dan Karakterisasi α-amilase Termostabil dari Bacillus stearothermophilus TII-12

Jurnal AgroBiogen ◽

10.21082/jbio.v7n1.2011.p56-62 ◽

2011 ◽

Vol 7 (1) ◽

pp. 56 ◽

Cited By ~ 1

Author(s):

Puji Lestari ◽

Nur Richana ◽

Abdul Aziz Darwis ◽

Khaswar Syamsu ◽

Untung Murdiyatmo

Keyword(s):

Bacillus Stearothermophilus ◽

Specific Activity ◽

Gel Filtration ◽

Total Activity ◽

Purification And Characterization ◽

Food Industries ◽

High Temperature Process ◽

Hydrolysis Products ◽

Starch Liquefaction

Purification and Characterization of Thermostable α-amylase from Bacillus stearothermophilus TII-12. Puji Lestari, Nur Richana, Abdul A. Darwis, Khaswar Syamsu, and Untung Murdiyatmo. Thermostable α-amylase is a potential enzyme employed in the starch processing and widely used in food industries, but this enzyme is still imported. The local enzyme production would be more economist and useful for its broad applications. Here we report α-amylase from indigenous bacteria TII-12 which was purified and characterized, as well as analyzed its hydrolysis product on cassava starch. The enzyme of Bacillus stearothermophilus TII-12 partially purified by ultrafiltration, acetone precipitation and gel filtration (Sephadex G-100) showed the reduced total activity, total protein and yield, but increased the specific activity. The enzyme had a Km of 1,06 mg/ml and Vmax of 1,21 mol/min, with optimal activity at pH 7 and 90oC. An apparent molecular mass was of 192.932,8 Dalton, as estimated by Native-Polyacrylamide Agarose Gel electrophoresis. Its activity was inhibited by the divalent cation chelator such as EDTA and CuSO4 but activated by calcium ion. Hydrolysis products of this enzyme on cassava starch were glucose, dextrin, maltose and oligosaccharides. After 24 hours of hydrolysis, the concentration of glucose and maltose reached 51.970 and 10.090 ppm, respectively. The thermostable α-amylase of TII-12 is an endo-α-amylase and prospective to be applied on starch liquefaction with high temperature process.

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