• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1532-1539
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• 2012

The ${\alpha}$-galactosidase-coding gene agaAJB13 was cloned from Sphingomonas sp. JB13 showing 16S rDNA (1,343 bp) identities of ${\leq}97.2%$ with other identified Sphingomonas strains. agaAJB13 (2,217 bp; 64.9% GC content) encodes a 738-residue polypeptide (AgaAJB13) with a calculated mass of 82.3 kDa. AgaAJB13 showed the highest identity of 61.4% with the putative glycosyl hydrolase family 36 ${\alpha}$-galactosidase from Granulicella mallensis MP5ACTX8 (EFI56085). AgaAJB13 also showed <37% identities with reported protease-resistant or Sphingomonas ${\alpha}$-galactosidases. A sequence analysis revealed different catalytic motifs between reported Sphingomonas ${\alpha}$-galactosidases (KXD and RXXXD) and AgaAJB13 (KWD and SDXXDXXXR). Recombinant AgaAJB13 (rAgaAJB13) was expressed in Escherichia coli BL21 (DE3). The purified rAgaAJB13 was characterized using p-nitrophenyl-${\alpha}$-D-galactopyranoside as the substrate and showed an apparent optimum at pH 5.0 and $60^{\circ}C$ and strong resistance to trypsin and proteinase K digestion. Compared with reported proteaseresistant ${\alpha}$-galactosidases showing thermolability at $50^{\circ}C$ or $60^{\circ}C$ and specific activities of <71 U/mg with or without protease treatments, rAgaAJB13 exhibited a better thermal stability (half-life of >60 min at $60^{\circ}C$) and higher specific activities (225.0-256.5 U/mg). These sequence and enzymatic properties suggest AgaAJB13 is the first identified and characterized Sphingomonas ${\alpha}$-galactosidase, and shows novel protease resistance with a potential value for basic research and industrial applications.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.27 no.1
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• pp.60-65
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• 1982

In order to observe the degree and response of drought-resistance and its physiological mechanism in barley and wheat seedling stage, 5 species (16 cultivars) were tested for the changes of nitrate reductase and protease activity and the accumulation of free proline, by being subjected to water stress by withholding watering for 8 days at 10 days (at the 3rd leaf stage) after emergence and by imposing water stress to the excised first leaf by polyethyleneglycol solution (osmotic potential, -20 bars) for 48 hours. The average rate of decrease of all cultivars was 42% in nitrate reductase activity and 73% in protease activity. But proline content in water stress was increased 10 folds more than that of control. The decrease4 rate of nitrate reductase activity in 5 species was in the order of wheat < rye < covered barley < naked barley < two-row barley: wheat being the lowest. The decreased rate of protease activity in 5 species was in the order of wheat > rye > two-row barley > covered barley > naked barley: wheat being the" heighest. The accumulated amount of free proline in 5 species by water stress was in the order of wheat > covered barley > rye > naked barley > two-row barley. And the increased ratio (folds) of free proline of water stress to control was in the order of rye(13) > wheat. covered barley(11) > naked barley(99) > two-row barley(7): rye being the highest. In terms of the enzymatic activity and the physiotically adaptive metabolism during the processing leading to drought-resistance, the degree of drought-resistance of 5 species to water stress at seedling stage was shown to be in the order of wheat > rye > covered barley > naked barley > two-row barley.

• Current Research on Agriculture and Life Sciences
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• v.32 no.3
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• pp.149-154
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• 2014

It is already known that adzuki beans (Vigna angularis) are able to control appetite. Therefore, this study tested the proteins isolated from adzuki beans for their protease resistance and interaction with the intestinal mucosa. The major proteins from adzuki beans were found to be resistant to the digestive enzymes pepsin and pancreatin, and were identified using 2D-SDS-polyacrylamide gel electrophoresis and mass spectrometry. The major adzuki proteins were easily fractionated by treating the soluble protein extract with 10mM $CaCl_2$, and were found to contain lactotransferrin, a homologous protein to the dynein light chain domain, proteinase inhibitor, and proteins with unknown functions. From a tissue binding assay using mouse intestinal tissue sections, the major protein fraction showed weak, yet significant and specific binding to the mucosa layer of the small intestine. Thus, the current results suggest that adzuki proteins are resistant to digestive enzymes, which enables them to survive protease digestion in the intestinal tract, plus they may interact with the intestinal mucosa layer. Therefore, the molecules responsible for controlling appetite in adzuki beans are presumably protease-resistant proteins that interact with the intestinal mucosa or delay digestion in the digestive tract.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.402-405
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• 2005

We have previously identified genes for four different protease inhibitors (PIs) that were induced upon rice blast infection in a rice blast resistant mutant SHM-11. Our expression analysis of the PIs indicated that induction of the PIs was the highest 24 hr after rice blast inoculation in the rice mutant SHM-11. Three PIs in the group of serine PIs were highly expressed while a cystein PI was weakly expressed upon rice blast inoculation. Four PIs were weakly induced 48 hr after pathogen inoculation in rice blast susceptible wild type rice plant. The simultaneous expression of three serine PIs was apparent from SHM-11 and two of them were induced in rice blast resistant Taebaegbyeo. One of them was induced in rice blast resistant Hwayeongbyeo while none of them were expressed in rice blast susceptible Nagdongbyeo and rice blast resistant Dongjinbyeo. Our results suggest that the expression of PI gene is rice cultivar specific and may be linked with the rice blast resistance in a specific rice mutant by the simultaneous expression of the PI genes.

• Journal of Microbiology
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• v.33 no.4
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• pp.344-349
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• 1995

The alkalophilic bacterium, Xanthomonas sp. JK311, producing extracellular proteases, was isolated from soil. Xanthomonas sp. JK311 produced five extracellular proteases that are all metalloproteases. Four of them were resistant against 1% SDS. Chromosomal DNA of the Xanthomonas sp. JK311 was digested with BamHI and cloned into PUC19. Among E. coli strain HB101 transformants, a clone secreting the proteases was screened through halo formation on skim-milk agar plate and by Southern blot analysis. It had the recombinant plasmid pXEP-1 containing the 7.5 kb-BamHI DNA fragment and produced three extacellular proteases. Their protease properties corresponded to those of Xanthomonas sp. JK311.

• Microbiology and Biotechnology Letters
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• v.7 no.3
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• pp.157-164
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• 1979

These experiments were performed to investigate the culture condition, characteristic of crude enzyme and the heat resistance of the acid protease by Aspergillus awamori U-3. The results obtained were as follows: 1. The optimum culture temperature and time on wheat bran medium and defatted rice bran medium were 3$0^{\circ}C$ and 72 hrs, respectively. The optimum amount of added water was 100~120 ％ on wheat bran medium and 100~130 ％ on de-fatted rice bran medium. 2. Of the these various ingredients, addition of KN $O_3$, glutamic acid and glucose on wheat bran medium and addition Of KN $O_3$, (N $H_4$)$_2$S $O_4$, glucose, lactose, K $H_2$P $O_4$ and MgC $l_2$ on defatted rice bran medium were very effective. On wheat bran medium, concentration of addition of glucose, KN $O_3$ and glutamic acid were 3.0~4.0％, 0.2~0.4 ％ and 1.0％, respectively. 3. The optimum pH for the enzyme action was 2.4 ％, the optimum temperature about 45$^{\circ}C$ and the stable pH range 2.0~5.0, The enzyme was stable below 5$0^{\circ}C$ and was inactivated rapidly above 5$0^{\circ}C$. 4. The addition of CaC $l_2$ and CaS $O_4$ as the heat resistance agents showed the slight resistance. 5. When the enzyme solution added with the heat resistance agents (CaC1$_{2}$ and CaS $O_2$) was heated for 10-30 minutes at 6$0^{\circ}C$, their remaining activities were decreased largely above 20 minutes and The heat resistance effects of CaC $l_2$ and CaS $O_4$ were not observed almost at 8$0^{\circ}C$.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.219-219
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• 2005

• The Journal of Korean Society of Virology
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• v.28 no.4
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• pp.295-302
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• 1998

The RNA genome of poliovirus encodes a long polyprotein precursor and this polyprotein is cleaved proteolytically by viral protease to yield mature proteins. The mature proteins derived from the P1 polyprotein precursor are the component of capsids. To further delineate the process of capsid assembly and encapsidation, in a first attempt, a cell line which expresses the authentic P1 polyprotein was established. CV-1 cells were transfected with the pRCRSVS1P1 plasmid DNA which contains 5'ncr sequences, whole authentic capsid gene of poliovirus and neomycin resistance gene. These cells were treated with G418 for 3 months, and eventually G418 resistant cells were selected and formed colonies. Each colony was picked and grown in the media containing G418. DNA analysis indicated that 1 of 13 neomycin resistant cell lines (R2-18) contains whole poliovirus P1 capsid gene segment which was incorporated into the genome. Immuneprecipitation of cell lysates with sera from rabbit immunized with inactivateded Sabin type 1 particles demonstrated the constitutive expression of the poliovirus P1 capsid protein from R2-18.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.180-184
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• 2003

Several bacteria and yeasts were isolated from soil and characterized for the development of functional probiotics which can be used as a livestock feed additive. From the soil, the microbial strains which have acid/bovine resistance, antibiotics resistance and high stability, were isolated. Most strains selected were very tolerable against acids and very stable in a broad range of pH. Some strains could survive 100% at pH 2.5. The growth of the strains was not affected in the presence of bile acid, pathogenic E. coli and several antibiotics such as tetracycline, nisin, kanamycin, streptomycin, ampicillin. Acidogenic capability test showed that all the strains can produce acids. The hydrolytic activities were analysed for amylase, protease, lipase and cellulase to decompose various organic compounds. All the strains were found to be gram negative, round type, non-kinetic and the color is yellow or white.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.12
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• pp.1822-1829
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• 2003

Porcine colostrum and milk were separated into the acid-soluble and casein fractions by acidification followed by centrifuge. The acid-soluble fraction of porcine colostrum was further separated by liquid chromatography and anisotropic membrane filtration. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and their components was determined by incubating bovine trypsin or chymotrypsin in a medium containing their corresponding substrates with or without addition of various amounts of porcine colostrum, porcine milk or their components. The inhibition of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) degradation in pig small intestinal contents by porcine colostrum was measured by incubating iodinated IGF-I or EGF with the intestinal contents with or without addition of porcine colostrum. Degradation of labeled IGF-I or EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid (TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory activity and increased the stability of IGF-I and EGF in pig intestinal contents. The inhibitory activity declined rapidly during lactation. It was also found that trypsin and chymotrypsin inhibitory activity and the inhibition on IGF-I and EGF degradation in the acid-soluble fraction were higher than that in the casein fraction. Heat-resistance study indicated that trypsin inhibitors in porcine colostrum survived heat treatments of $100^{\circ}C$ water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heatsensitive. Separation of the acid-soluble fraction of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the trypsin and chymotrypsin inhibitory capacity was mainly due to a group of small proteins with molecular weight of 10,000-50,000. In conclusion, the present study confirmed the existence of high levels of protease inhibitors in porcine colostrum, and the inhibition of porcine colostrum on degradation of milk-borne growth factors in the pig small intestinal tract was demonstrated for the first time.