Purification and characterization of two isoforms of native α amylase from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong)

2017 ◽  
Vol 42 (6) ◽  
Author(s):  
Raksmont Ubonbal ◽  
Saijai Porsoongnoen ◽  
Jureerut Daduang ◽  
Sompong Klaynongsruang ◽  
Sakda Daduang

AbstractIntroduction:The tropical plant amylases involved in the fruit ripening stage is outstanding for their high activities in converting starch to sugars within a short period at high temperatures over 40°C.Methods:The α amylase iso-enzymes from Ok-Rong mango (Results:The enzyme was purified 105-fold with a final specific activity of 59.27 U mgConclusion:Two α amylase iso-enzymes were classified as members of the low-pI group of amylases with identical structure, properties and functions. They are mesophilic with high possibilities for application for many purposes.

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.


1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf

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.


1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.


1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.


1981 ◽  
Vol 197 (2) ◽  
pp. 413-419 ◽  
Author(s):  
P F Leadlay

Methylmalonyl-CoA epimerase, which specifically interconverts the (2R)- and (2S)- epimers of methylmalonyl-CoA, was purified 95-fold from Propionibacterium shermanii by a new method that affords apparently homogeneous enzyme, in 80-100mg quantities, in yields representing about 40% of the activity in cell-free extracts. The specific activity of the purified enzyme, 10.1 mukat/mg, is much greater than previously reported. Native methylmalonyl-CoA epimerase has Mr about 33000, and apparently consists of two identical subunits. The purified enzyme is stable indefinitely when stored at -20 degrees C and pH 8.5, but contrary to previous reports it is not unusually acid-stable. The activity of methylmalonyl-CoA epimerase is increased by Co2+, and to a smaller extent by Ni2+, Mn2+ and Zn2+.


Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7545
Author(s):  
Jianyou Zhang ◽  
Guangcheng Zhou ◽  
Lifeng Fei ◽  
Lifan Chen ◽  
Lei Sun ◽  
...  

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.


Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599
Author(s):  
J Loscalzo ◽  
J Freedman

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.


Author(s):  
Chu Thanh Binh ◽  
Nguyen Phuong Nhue ◽  
Ho Tuyen ◽  
Bui Thi Viet Ha

The nematophagous – fungi Paecilomyces sp. is curently developed as a biocontrol agent against plant parasitic nematodes (Khan et al., 2003; Yang et al., 2007). Biological control agents can infiltrate certain nematode sites and destroy them by producing some enzymes including chitinase (Khadijeh et al., 2017). The purpose of this study was to purify, determine the chitinase activity from Paecilomyces sp. P1. With Lugol reagent, chitinase of this strain was characterized by diffusion on agar plate. Chitinase specific activity was determined by measuring the release of reducing saccharides from colloidal chitin by the N-acetyl-glucosamine-dinitrosalicylate method at 540 nm. By using the saturated (NH4)2SO4 precipitation at 65% concentration, DEAE A-50 ion exchange chromatography and SDS - PAGE concentration 12.5%, chitinase molecules weigh nearly 50kDa, having a specific activity of 133,3 U/mg, 2,1-fold higher than that of supernatant. Furthermore, method of testing with the nematode Meloidogyne sp., the ability to kill nematodes of Paecilomyces sp. P1 reached 58% efficiency in 96h. These results were a scientific basis for the application of Paecilomyces sp. P1 in the production of nematode insecticides. Keywords Paecilomyces sp. P1; chitinase; purify, biocontrol, Meloidogyne sp References   [1] Nguyễn Ngọc Châu, Tuyến trùng thực vật và cơ sở phòng trừ, NXBKHKTHN, 2003.[2] Nguyễn Hữu Quân, Vũ Văn Hạnh, Quyền Đình Thi, Phạm Thị Huyền, Tinh sạch và đánh giá tính chất lý hóa của chitinase từ nấm Lecanicillium lecanii, Kỷ yếu Hội nghị Công nghệ Sinh học toàn quốc, 1 (2013) 426.[3] CM Baratto, V Dutra, JT Boldo, LB Leiria, MH Vainstein, A. Schrank Isolation, characterization and transcriptional analysis of the chitinase chi2 gene (DQ011663) from the biocontrol fungus Metarhizium anisopliae var. anisopliae., Curr Microbiol, 53 (2006) 217.[4] D. Wharton,. Nematode eggshells, Parasitology 81 (1980) 447.[5] F. A. Zaki, D. S. Bhatti , Effect of castor (Ricinus communus) and the biocontrol fungus Paecilomyces lilacinus on Meloidogyne javanica, Nematologica 36 (1980) 114.[6] H. M. Hussein Al Ajrami., Evaluation the Effect of Paecilomyces lilacinus as a Biocontrol Agent of Meloidogyne javanica on Tomato in Gaza Strip, Faculty of science Master of Biological Sciences Microbiology., 2016.[7] J. De la Cruz, A Hidalgo-Gallego, JM Lora, T Benitez, JA Pintor-Toro, A Llobell , Isolation and characterization of three chitinases from Trichoderma harzianum., Eur. J. Biochem,. 206 (1992) 859.[8] JLD Marco, MC Valadares-Inglis . Purification and characterization of an N-acetylglucosaminidase produced by a Trichodermaharzianum strain which controls Crinipellis perniciosa. Appl. Microbiol. Biotechnol. 64 (2003) 70.[9] JLD Marco , LHC Lima, MV Sousa MV, CR Felix A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa, the causal agent of witches’ broomof cocoa, J Microbiol Biotechnol 16 (2000) 383.[10] Khan Alamgir, Williams Keith, Mark P. Molloy, and Nevalainen Henlena, Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels, Protein Expression and Purification 32 (2003) 210.[11] Khadijeh Abbsi, Doustmorad ZAFARI, Robert WICK., Evaluation of chitinase enzyme in fungal isolates obtained from golden potato cyst nematode (Globodera rostochiensis) Zemdirbyste-Agriculture, 2 (2017) 179.[12] Kopparapu Narasimha Kumar, Peng Zhou, Shuping Zhang, Qiaojuan Yan, Zhuqing Liu, Zhengqiang Jiang, Purification and characterization of a novel chitinase gene from Paecilomyces thermophila expressed in Escherichia coli. Carbonhydrate Reseach 347 (2012) 155.[13] Methanee Homthong, Anchanee Kubera, Matana Srihuttagum, Vipa Hongtrakul, Isolation and characterization of chitinase from soil fungi, Paecilomyces sp. Agriculture and Natural Resources, 1 (2016) 50.[14] RS Patil, V Ghormade, MV Desphande MV ,Chitinolytic enzymes: an exploration. Enzyme Microb. Technol. 26 (2002) 473[15] RJ Leger St , RM Cooper, AK Charnley, Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 58 (1991) 415.[16] S Leger, RJ Joshi RJ, RJ Bidochka, DW Roberts . Characterization and ultrastructural localization of Metarhizium anisopliae, M. xavoviride, and Beauveria bassiana during fungal invasion of host (Manduca sexta) cuticle. Appl Environ Microbiol 62 (1996)907.[17] SC Kang, S. Park, DG Lee ,, Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhiziumanisopliae. J Invertebr Pathol., 73 (1999) 276.[18] P.J.M Bonants, P.F.L. Fitters, H. Thijs, E. den Belder, C. Waalwijk, J.W.D.M. Henfling. A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs, Microbiology 141(1995) 75.[19] VE Tikhonov, LV Lopez-Llorca, J Salinas, HB Jansson . Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium, Fungal Genet Biol (2002) 67[20] Van Nam Nguyen, YJ Kim, KT Oh, WJ Jung, RD Park , The antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9. Curr. Microbiol 56 (2008) 28.[21] Van Nam Nguyen, In-Jae Oh, Young-Ju Kim, Kil-Yong Kim, Young-Cheol Kim, Ro-Dong Par J Ind., Purification and characterization of chitinases from Paecilomyces variotii DG-3 parasitizing on Meloidogyne incognita eggs, (2009) 195[22] Z. Perveen and S. Shahzad S., , A comparative study of the efficacy of Paecilomyces species against root-knot nematode Meloidogyne incognita. Pakistan Journal of Nematology, 31 (2013) 125


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