ABSTRACT
Lipase was partially purified from the culture supernatant of Mucor racemosus NRRL 3631. In an attempt to increase the enzyme stability, the enzyme was immobilized on poly (vinyl alcohol) PVA, radiation cross liked poly (vinyl alcohol/ vinyl pyrrolidone) PVA / PVP and poly (vinyl alcohol/ hydroxyethylmethacrylate) PVA/ HEMA hydrogels. The maximum immobilization yield (31.74 %) was obtained using PVA/ HEMA copolymer. The effect of the immobilization parameters on the enzyme such as the hydrogel composition, irradiation dose and the immobilization technique was performed. An optimum radiation dose of 15 kGy and hydrogel composition of 10 % PVA/ HEMA (9.6: 0.4 v/v) increased the immobilization yield to 60.3 %. Diffusion phenomena can be markedly increased the enzyme immobilization on the surface of the hydrogel. In this case the retained activity was approximately 81.5 % of that of the free enzyme. The profiles of immobilized enzyme activities at various pH values (4-9) and temperatures (30-80 °C) showed an overall higher stability for the immobilized enzyme than that for the free one. The half life values of the immobilized and free enzymes at 60 °C were 3.3 h and 1.73 h, respectively. The immobilized enzyme retained 69.2 % of its initial activity after three cycles.
ABSTRACT
The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73 percent) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.
Subject(s)
/genetics , DNA, Complementary/genetics , Escherichia coli/genetics , Genetic Vectors/genetics , Mucor/enzymology , Base Sequence , Cloning, Molecular , /metabolism , Gene Library , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Open Reading Frames , Transcription, GeneticABSTRACT
A soft rot of fruits caused by Mucor racemosus occurred on cherry tomato collected in Agricultural Products Wholesale Market in Jinju, Korea. The disease infection usually occurred wounded areas after cracking of fruits. At first, the lesions started with water soaked and rapidly softened and diseased lesion gradually expanded. Colonies were white to brownish to gray in color. Sporangia were 32~54 microm in size and globose in shape. Sporangiophores were 8~14 microm in width. Sporangiospores were 5~12 x 4~8 microm in size, ellipsoidal to subglobose in shape. Columella was 27~42 microm in size, obovoid, ellipsoidal, cylindrical-ellipsoidal, slightly pyriform in shape. Chlamydospores were numerous in sporangiophores and barrelshaped when young, subglobose in old cultures. Optimum growth temperature was about 25degrees C. The fungus was identified as M. racemosus Fres. This is the first report of soft rot on cherry tomato caused by M. racemosus in Korea.