Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Structure and activity of angiotensin I converting enzyme inhibitory peptides derived from Alaskan pollack skin

Angiotensin I that converts the enzyme (ACE) inhibitory peptide, Gly-Pro-Leu, previously purified and identified from the Alaskan pollack skin gelatin hydrolysate, were synthesized. In addition, the peptides Gly-Leu-Pro, Leu- Gly-Pro, Leu-Pro-Gly, Pro-Gly-Leu, Pro-Leu-Gly, Gly- Pro, and Pro-Leu, which consisted of glycine, proline, and leucine, were synthesized by the solid-phase method. The IC50 values of each tripeptide. namely Leu-Gly-Pro, Gly- Leu-Pro, Gly-Pro-Leu, Pro-Leu-Gly, Leu-Pro-Gly, and Pro-Gly-Leu. were 0.72, 1.62, 2.65, 4.74, 5.73, and 13.93 microM, respectively. The ACE inhibitory activity of these tripeptides was higher than that of dipeptides, such as Gly- Pro and Pro-Leu with IC50 values of 252.6 and 337.3 microM, respectively. Among the tripeptides, Leu-Gly-Pro and Gly- Leu-Pro had higher inhibitory activity than Gly-Pro-Leu that was isolated from the Alaskan pollack skin gelatin hydrolysate. Among the different types of tripeptides that were examined, the highest ACE inhibitory activity was observed for Leu-Gly-Pro. It had the leucine residue at the N-terminal and proline residue at the C-terminal.     

An analysis of turbulent shear stresses in leakage flow through a bileaflet mechanical prostheses

In this work, estimates of turbulence were made from pulsatile flow laser Doppler velocimetry measurements using traditional phase averaging and averaging after the removal of cyclic variation. These estimates were compared with estimates obtained from steady leakage flow LDV measurements and an analytical method. The results of these studies indicate that leakage jets which are free and planar in shape may be more unstable than other leakage jets, and that cyclic variation does not cause a gross overestimation of the Reynolds stresses at large distances from the leakage jet orifice.     

A novel amylolytic enzyme from Thermotoga maritima,resembling cyclodextrinase and alpha-glucosidase,that liberates glucose from the reducing end of the substrates

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.     

Cloning of a ribonucleotide reductase gene of the herpes simplex virus type 2 strain G

The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.     

Identification of a flower-specific cDNA,<Emphasis Type="Italic">RsPCP1</Emphasis>, encoding putative pollen coat protein from radish

To better understand the molecular control of floral development, we identified a flower-specific cDNA,RsPCPI, from Korean radish (Raphanus sativus). Based on nucleotide sequence analysis, this clone contains an open reading frame of 65 amino acids and shares 91% identity with a pollen coat protein from cabbage (Brassica oleracea). Southern analysis revealed thatRsPCPI is present as a single-copy gene or a member of a small gene family in the radish genome. BecauseRsPCPI mRNA was present exclusively in mature floral buds but not in young floral buds or in vegetative tissues, we propose that this gene is anther-specific.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

Novel BOD (biological oxygen demand) sensor using mediator-less microbial fuel cell

A microbial fuel cell type of biosensor was used to determine the biochemical oxygen demand (BOD) of wastewater. The biosensor gave a good correlation between the BOD value and the coulomb produced. The BOD sensor has been operated for over 5 years in a stable manner without any servicing. This is much longer that that of previously reported BOD biosensors.