ABSTRACT
Two ustilaginomycetous anamorphic strains were isolated from flowers in Taiwan. Phylogenetic analysis based on the combined rRNA gene sequence of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 and large-subunit D1/D2 domains indicated that the closest recognized species was Pseudozyma fusiformata. The results of DNA-DNA hybridization and physiological characteristics showed that the two strains represent a novel species within the genus Pseudozyma. The name Pseudozyma pruni sp. nov. is proposed, with FIRDI 005(T) (=BCRC 34227(T) =CBS 10937(T)) as the type strain.
Subject(s)
Flowers/microbiology , Prunus/microbiology , Ustilaginales/classification , DNA, Fungal/analysis , DNA, Ribosomal Spacer/analysis , Genes, rRNA , Genotype , Molecular Sequence Data , Mycological Typing Techniques , Nucleic Acid Hybridization , Phenotype , Phylogeny , RNA, Ribosomal, 5.8S/genetics , Sequence Analysis, DNA , Species Specificity , Taiwan , Ustilaginales/genetics , Ustilaginales/isolation & purification , Ustilaginales/physiologyABSTRACT
Recombinant Streptomyces platensis transglutaminase (MtgA) produced by the Streptomyces lividans transformant 25-2 was purified by ammonium sulfate fractionation, followed by CM-Sepharose CL-6B fast flow, and blue-Sepharose fast flow chromatography. The purification factor was approximately 33.2-fold, and the yield was 65%. The molecular weight of the purified recombinant MtgA was 40.0 KDa as estimated by SDS-PAGE. The optimal pH and the temperature for the enzyme activity were 6.0 and 55 degrees C, respectively, and the enzyme was stable at pH 5.0-6.0 and at temperature 45-55 degrees C. Enzyme activity was not affected by Ca(2+), Li(+), Mn(2+), Na(+), Fe(3+), K(+), Mg(2+), Al(3+), Ba(2+), Co(2+), EDTA, or IAA but was inhibited by Fe(2+), Pb(2+), Zn(2+), Cu(2+), Hg(2+), PCMB, NEM, and PMSF. Optimization of the fermentation medium resulted in a twofold increase of recombinant MtgA activity in both flasks (5.78 U/ml) and 5-l fermenters (5.39 U/ml). Large-scale productions of the recombinant MtgA in a 30-l air-lift fermenter and a 250-l stirred-tank fermenter were fulfilled with maximal activities of 5.36 and 2.54 U/ml, respectively.
Subject(s)
Industrial Microbiology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Streptomycetaceae/enzymology , Transglutaminases/biosynthesis , Transglutaminases/genetics , Cations/pharmacology , Culture Media , Hydrogen-Ion Concentration , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Streptomyces lividans/drug effects , Streptomyces lividans/enzymology , Streptomyces lividans/genetics , Streptomycetaceae/genetics , Temperature , Thermodynamics , Time Factors , Transglutaminases/isolation & purification , Transglutaminases/metabolismABSTRACT
An efficient system for the fast and efficient purification of transglutaminase from recombinant Streptomyces platensis and expressed in Streptomyces lividans 25-2 is described. Because the purification procedure of this system is flexible, culture broth from laboratory (20 l) and pilot-plant (130 l) fermentations were used to purify the enzyme to electrophoretic homogeneity with high purity (90-95%) and yield (61-77%) within 1 or 2 days.
Subject(s)
Chemical Fractionation/methods , Genetic Enhancement/methods , Streptomyces/enzymology , Streptomyces/metabolism , Transglutaminases/chemistry , Transglutaminases/isolation & purification , Pilot Projects , Recombination, Genetic/genetics , Transglutaminases/genetics , Transglutaminases/metabolismABSTRACT
Aqualysin I has at least two Ca2+-binding sites that have different affinities for Ca2+. The binding of various metal ions to aqualysin I was studied using 23Na- and 139La-NMR spectrometry. Evidence is presented that Ca2+, La3+, and Na+ bind to the low-affinity Ca2+-binding site of aqualysin I, but Mg2+ does not. Our results confirm that binding of metals at the low-affinity Ca2+-binding site is essential for thermostabilization, since the addition of Mg2+ did not result in thermostabilization. La3+ was found to bind to both the low-affinity Ca2+-binding site and an additional metal ion-binding site that can also be involved in the thermostabilization of aqualysin I.
