• Korean Journal of Food Science and Technology
• /
• v.43 no.6
• /
• pp.729-734
• /
• 2011

With the goal of transforming rice protein from an insoluble to a soluble form to increase the industrial utilization of rice wine meal (RWM), RWM was derivatized using commercial proteases and the RWM hydrolysates were characterized. Eight commercial proteases were used individually or in combination for hydrolysis of RWM. The degree of hydrolysis was assessed by determining the soluble protein in supernatant using the Lowry assay, protein in precipitates using a semimicro Kjeldahl procedure, and gravimetrically by the weight difference before and after hydrolysis. Protamex, Alcalase and Protease N proteases were most effective for hydrolysis of RWM. Although these assessment methodologies displayed some variation, they generally showed a similar pattern. When the aforementioned three proteases were simultaneously used to treat RWM, no significant difference was observed between the three assays (p<0.05) indicating an absence of enzymatic synergy.

• Journal of the Korean Magnetic Resonance Society
• /
• v.19 no.2
• /
• pp.74-82
• /
• 2015

Ryanodine receptor (RyR) is one of the two major $Ca^{2+}$ channels in membranes of intracellular $Ca^{2+}$ stores and is found in sarcoplasmic reticulum (SR), endoplasmic reticulum (ER). RyR1 is also the major calmodulin-binding protein of sarcoplasmic reticulum membranes. Residues 4064-4210 in the RyR1 polypeptide chain has similar primary sequence with calmodulin (CaM) and was designated as CaM-like domain (CaMLD). When expressed as a recombinant peptide, CaMLD showed several CaM-like properties in previous studies. Still, previous studies of CaMLD were focused on protein-protein interactions rather than its own properties. Here, we studied the expression of CaMLD and its sub-domains corresponding to each lobe of CaM in Escherichia coli. CaMLD could be obtained only as inclusion body, and it was refolded using urea solubilization followed by dialysis. Using spectroscopic approaches, such as NMR, circular dichroism, and gel filtration experiment, we found that the refolded CaMLD exists as nonspecific aggregate, even though it has alpha helical secondary structure. In comparison, the first half of CaMLD (R4061-4141) could be obtained as natively soluble protein with thioredoxin fusion. After the removal of the fusion tag, it exhibited folded and helical properties as shown by NMR and circular dichroism experiments. Its oligomeric status was different from CaMLD, existing as dimeric form in solution. However, the second half of the protein could not be obtained as soluble protein regardless of fusion tag. Based on these results, we believe that CaMLD, although similar to CaM in sequence, has quite different physicochemical properties and that the second half of the protein renders it the aggregative properties.

• BMB Reports
• /
• v.37 no.4
• /
• pp.408-415
• /
• 2004

The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and $40^{\circ}C$. The catalytic efficiency ($k_{cat}/K_m$) values for $\alpha$-, $\beta$-, and $\gamma$-CD were $3.0{\times}10^5$, $8.8{\times}10^5$, and $5.5{\times}10^5\;M^{-1}\;min^{-1}$, respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.

• Parasites, Hosts and Diseases
• /
• v.39 no.2
• /
• pp.119-132
• /
• 2001

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of Dl fraction (DIA) among 5 antigenic protein fractions partially purified by DEAE- anion exchange chromatography from water- soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. DlA showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that DlA was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immune-reactivity of two immunoglobulins (PI-Ig, Dl-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with Dl-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in Dl antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue .

• Journal of Microbiology and Biotechnology
• /
• v.10 no.2
• /
• pp.221-226
• /
• 2000

Cephalosporin-C deacetylase (CAH) catalyzes the deacetylation of cephalosporin derivatives. A novel gene encoding the CAH from Bacillus sp. KCCM10143 was cloned and sepuenced. The uncleotide sequence contained an open reading frame encoding a polypeptide consisting of 217 amino acids and a molecular weight of 24 kDa which was in good agreement with the value obtained by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An expression plasmid was constructed by inserting the CAH gene into the region of the pTrc99A expression vector. An active from of the CAH protein was expressed in the soluble fraction obtained after cell disruption. in fermentation using a 5-1 jar fementer, the transformant E. coli JM109 (pDST654) produced 4.12 U of CAH per ml of culture during 16 h of incubation.

• Korean Journal of Microbiology
• /
• v.27 no.3
• /
• pp.237-244
• /
• 1989

A soluble intracellular protease from cells of Pseudomonas carboxydovorans, a carboxydobacterium, grown on nutrient broth was purified 68-fold in five steps to better than 95% homogeneity with a yield of 2.4% using azocasein as a substrate. The enzyme activity was not detected from cells grown on pyruvate, succinate, acetate, or CO as a sole source of carbon and energy. The molecular weight of the native enzyme was determined to be 53,000. Sodium dodecyl sulfate-gel electrophoresis revealed the purified enzyme a monomer. The enzyme was found to be a serine-type protease. The enzyme activity was inhibited completely by several divalent cations such as $Cd^{2+}, Cu^{2+}, Hg^{2+}$, and $Fe^{2+}$. The enzyme was also inhibited by EGTA, but was stimulated by iodoacetamide. The optimal pH and temperature for the enzyme reaction were found to be 8.0 and $50^{\circ}C$, respectively. The enzyme was inactive on CO dehydrogenase.

• BMB Reports
• /
• v.37 no.2
• /
• pp.159-166
• /
• 2004

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of $3.4{\times}10^{-7}\;M$. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.

• Journal of Life Science
• /
• v.12 no.6
• /
• pp.688-693
• /
• 2002

Molecular chaperones prevent the misfolding of newly synthesized polypeptides in the cell. The coexpression of molecular chaperones could be expected to improve the production of soluble and active recombinant proteins. In this study, the effect of coexpression of E. coli GroEL/ES chaperone on the active production of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in E. coli was investigated. Two plasmids, pTCGT1 and pGro7 in which the cgt and the groEL/ES genes are under the control of 77 promoter and araB promoter, respectively, were co-transformed into E. coli. With a series of cultures of recombinant E. coli cells, the optimal concentrations of IPTG and L-arabinose were found be 1 mM and 0.3 mg/$m\ell$, respectively. When IPTG and L-arabinose were added at 0.8~1.0 $OD_{600}$ and 0.4~0.5 $OD_{600}$, active CGTase production was increased significantly. This coexpression condition resulted in 1.5-fold increased level of soluble CGTase (0.7~0.73 unit/$m\ell$), compared to the level of CGTase in the single expression (0.36~0.56 unit/$m\ell$). An SDS-PACE analysis revealed that about 33.6% of CGTase in the total CGTase protein was found in the soluble fraction by coexpression of GroEL/ES chaperone.

• Korean Journal of Medicinal Crop Science
• /
• v.14 no.4
• /
• pp.195-201
• /
• 2006

The fibrinolytic activities of soluble proteins extracted from young leaves of Camellia japonica L. were studied. Fibrinolvity activity of extract from partitions of C. japonica L. showed 1.6-2.0 times higher than plasmin used as positive control. The fibrinolytic enzyme was confirmed directly from young leaves of C. japonica L. by a fibrin Plate and fibrin zymography. The protein was composed of a single polypeptide and its apparent molecular weight was found to be 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the fibrinolytic activity were pH 5.5 and $30^{\circ}C$, respectively. Also, the fibrinolytic activity was clearly inhibited by PMSF and TLCK, suggesting that it is a member of the trypsin-like serine protease. All these results suggest the protease is a fibrinolytic enzyme belong to a family of trypsin-like serine protease.

• KSBB Journal
• /
• v.21 no.1 s.96
• /
• pp.20-27
• /
• 2006

The fibrinolytic activities of soluble proteins extracted from seeds of Coix lacryma-jobi L., Carthamus tinctorius L. and Malva venicillata L. were studied. Fibrinolytic activity of extract from C. lacryma-jobi L. showed 1.3 times higher than plasmin used as positive control. The fibrinolytic enzyme was confirmed and extracted directly from seed of C. lacryma-jobi L. by a fibrin zymography. The protein was composed of a single polypeptide and its apparent molecular weight was found to be 7.8 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The effect of temperature for the proteolytic enzyme activity were stabilized above $50^{\circ}C$ and then dramatically decreased. Also, the enzyme activity was clearly inhibited by APMSF, PMSF and TPCK, suggesting that it is a member of the chymotrypsin-like serine pretense. In addition, effects of gamma-irradiated on seed of each plants were revealed that 8 Gy and 64 Gy were higher than others. This result shown that gamma-irradiation of seeds were capable to increase the fibrinolytic activity. All these results suggest the pretense is a fibrinolytic enzyme belong to a family of chymotrypsin-like serine pretense.