No abstract

Previously, we purified a strong fibrinolytic enzyme (subtilisin DJ‐4) from Bacillus sp. DJ‐4 and characterized its enzymatic activity. Here, we cloned the gene subtilisin DJ‐4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN′ from B. amyloliquefacens. Recombinant full‐subtilisin DJ‐4 (rf‐subDJ‐4) and mature‐subtilisin DJ‐4 (rm‐subDJ‐4) were expressed using a pET29 vector system, and their fibrin (ogen)olytic and plasminogen activator activities were studied. rf‐subDJ‐4 was found to have a higher stability to heat (60 °C) and to acidic conditions (pH 3.0–4.0) than the native subtilisin DJ‐4 of Bacillus sp. DJ‐4. The plasminogen activator activity of rf‐subDJ‐4 was 2.75 times greater than that of plasmin on a molar basis. And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN′ and subtilisin Carlsberg, respectively. rf‐subDJ‐4 rapidly hydrolyzed the Aα‐, Bβ‐, and γ‐chains of fibrinogen within 5 min. But, unlike subtilisin BPN′ at a very low concentration (50 ng), the γ‐chain was not cleaved. On the other hand, rm‐subDJ‐4 did not show enzyme activity.

A gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean fermented food. The L. lactis EG001 glucansucrase gene consists of 4503 bp open reading frame (ORF) and encodes an enzyme of 1500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 degrees C and pH 5.0. In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications.

A Gram-stain-negative, motile, rod-shaped, cellulose-degrading bacterial strain, BIO-TAS4-2 T , which belongs to the Betaproteobacteria, was isolated from forest soil from Naejang Mountain, Korea, and its taxonomic position was investigated by using a polyphasic study. Strain BIO-TAS4-2 T grew optimally at pH 7.0-8.0, at 30 6C and in the presence of 0-1.0 % (w/v) NaCl.Phylogenetic trees based on 16S rRNA gene sequences showed that strain BIO-TAS4-2 T clustered with members of the genera Andreprevotia, Silvimonas and Deefgea of the family Neisseriaceae, with which it exhibited 16S rRNA gene sequence similarities of 93.5-94.2 %. Strain BIO-TAS4-2 T contained Q-8 as the predominant ubiquinone and summed feature 3 (C 16 : 1 v7c and/or iso-C 15 : 0 2-OH) and C 16 : 0 as the major fatty acids. The DNA G+C content was 63.8 mol%. Strain BIO-TAS4-2 T could be differentiated from members of phylogenetically related genera by differences in fatty acid composition, DNA G+C content and some phenotypic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BIO-TAS4-2 T is considered to represent a novel species in a new genus, for which the name Jeongeupia naejangsanensis gen. nov., sp. nov. is proposed, with BIO-TAS4-2 T (5KCTC 22633 T 5CCUG 57610 T

A novel Wbrinolytic enzyme (AJ) was puriWed from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and Wbrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K m ) and K cat values of AJ towards -casein were 0.38 mM and 19.73 s ¡1 , respectively. AJ cleaved the A -chain of Wbrinogen but did not aVect the B -and -chains, indicating that it is an -Wbrinogenase. The Wbrinolytic activity was inhibited by diisopropyl Xuorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other Wbrinolytic enzymes.