• Journal of the Korean Wood Science and Technology
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• 제33권4호통권132호
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• pp.45-52
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• 2005

백색부후균 Phanerochaete chrysosporium으로부터 유래한 Manganese peroxidase (mnp5)를 methylotrophic yeast인 Pichia pastoris에서 이종 발현을 하였다. 이종발현으로부터 얻어진 단백질은 클로닝으로부터 예상되어지는 분자량보다 높은 분자량인 45 kDa으로 나타났다. 이것은 mnp5가 가지고 있는 glycosylation site에 의한 것이며, N-linked hyperglycosylation이 효소 활성에 영향을 미치는지를 site direct mutation에 의해 확인하였다. Sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)와 Coomassie Brilliant Blue (CBB) 염색에 의해 분자량을 확인한 결과 약 37 kDa으로 나타났으며, 효소활성을 측정한 결과 glycosylation이 효소 활성에 영향을 미치지 않는 것으로 나타났다. 따라서 본 연구로부터 P. pastoris에서 mnp5의 이종발현이 성공적으로 이루어졌으며 이러한 결과로부터 heme을 포함하고 있는 단백질의 이종발현 생산의 가능성을 보여주었다.

• Journal of Animal Science and Technology
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• 제62권5호
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• pp.741-752
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• 2020

Herein, the in vitro protein digestibility of lyophilized Protaetia brevitarsis larvae flour with and without defatting using 70% ethanol was compared with beef loin. Proximate analysis showed that the defatted larvae contained the highest protein content (p < 0.05). The viable counts of total aerobic bacteria, Escherichia coli, and coliform bacteria decreased significantly after defatting the larval samples with 70% ethanol (p < 0.05). Measurement of α-amino group content and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed higher amounts of low molecular weight proteins in the larvae compared to beef loin (p < 0.05). After in vitro digestion, the degree of protein hydrolysis of the digesta was higher for both larvae samples compared to beef loin (p < 0.05). No change was observed in the in vitro larval protein digestibility after defatting. These results highlight the excellent protein digestibility of P. brevitarsis larvae with high protein content. Defatting insect flour with 70% ethanol could enhance microbial safety while maintaining excellent protein digestibility.

• 한국축산식품학회지
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• 제38권5호
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• pp.950-958
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• 2018

The physicochemical characteristics and oxidative stability of wet-aged and dry-aged pork cuts were investigated at different aging periods (1, 7, 14 and 21 d). Samples were assigned into four groups in terms of shoulder blade-wet aging (SW), shoulder blade-dry aging (SD), belly-wet aging (BW), and belly-dry aging (BD). SD showed significantly higher pH at 21 d of aging than the other samples. Wet-aged cuts had significantly higher released water (RW) %, lightness ($L^*$) and shear force compared to the dry-aged meats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed greater degradation of proteins for dry-aged cuts than the wet-aged cuts. At the end of aging, wet-aged cuts showed significantly lower thiobarbituric acid-reactive substances (TBARS) value than the dry-aged samples, indicating higher oxidative stability for wet-aged pork cuts. However, dry-aging led to higher degradation of proteins resulting in increased water-holding capacity (WHC) and decreased shear force value.

• 한국미생물·생명공학회지
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• 제22권2호
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• pp.134-142
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• 1994

The neucleotide sequences of the xylA gene encoding $\beta $-xylosidase of Bacillus stearothermophilus and is its flanking regions were datermined. Three open reading frame(ORFs) were found, one of which(ORF1) appeared to code for the $\beta $-xylosidase. The 1830 base pair ORF1 encoded 609 amino acids starting from a TTG initiation codon. The molecular weight deduced from the nucleotide sequence(68 KD) was in agreement with that estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme(66 KD). The Shine-Dalgarno sequence(5'-AGGAGG-3') was found 11 bp upstream of the initiation codon. Further 15 bp upstream, there observed a potential transcription initiation signals. The putative -10 sequence(CATAAT) and -35 sequence(TTGTTA) coresponded closely to the consensus sequences for Bacillus subtilis RNA polymerase with major sigma factor. The guanine-plus-cytosine content of the coding region of the xylA gene was 56mol% while that of the third position of the codons was 63 mol%. Based on the comparison with the amino acid sequences of several other carbohydrate degrading enzymes, two conserved regions, possibly participating in the catalytic mechamism of $\beta $-xylosidase xylA, were identified in 278-298 and 329-350 regions of the translated xylA gene. The nucleotide sequence of the xylA was found to exhibit no homology to any other genes so far reproted.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.129-136
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• 1997

Helicobacter pylori is a spiral-shaped, microaerophilic human gastric pathogen causing chronic-active gastritis in association with duodenal ulcer and gastric cancer. To investigate the possibility of H. pylori outer membrane proteins (OMPS) as the oral vaccine antigens, sarcosine-insoluble outer membrane fraction has been prepared from H. pylori NCTC 11637. The major OMPs having apparent molecular masses of 62 kDa, 54 kDa and 33 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which were identified as urease B subunit (UreB), heat shock protein (Hsp54 kDa) and urease A subunit (UreA), respectively. Minor protein bands of 57 kDa, 52 kDa, 40 kDa, 36 kDa and 31 kDa were also observed. The antigenicity of H. pylori OMPs and antigenic cross-reactivity among the strains were determined by immunoblot analysis using anti-H. pylori OMPs antisera or intestinal lavage solutions. The results showed that UreB, Hsp54 kDa, UreA and 40 kDa proteins vigorously stimulated mucosal immune response rather than systemic immunity. From this results, these proteins seemed to be useful as the antigen candidates for the oral vaccine. The immunoblotting results with surface proteins from eight isolated H. pylori strains were similar to that of H. pylori NCTC 11637. The IgA which had been arised from oral administration of H. pylori OMPs, was able to bind H. pylori whole-cells.

• BMB Reports
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• 제30권4호
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• pp.274-279
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• 1997

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branchedchain amino acids, valine, leucine, and isoleucine. ALS is the target site for several structually diverse classes of herbicides including sulfonylureas, imidazolinones. and triazolopyrimidines. We have purified ALS from etiolated barley shoots to homogeneity. The five major purification steps are ammonium sulfate fractionation, DEAE anion exchange, hydroxylapatite, Bio-Gel A gel filtration, and low pressure Mono-Q chrornatoqraphy. Approximately 170-fold purification was achieved and the yield was 0.45% of initial activity in the crude extract. Both SDS-PAGE and Western blot analysis showed a single polypeptide of ALS with an apparent molecular mass of 64 kDa. The result of nondenaturing gel electrophoresis with activity staining indicated that the molecular mass of its native form is approximately 225 to 250 kDa. The values of $K_m$ for pyruvate. pl. and optimum pH of ALS were determined to be 2.0 mM, 5.2. and 7.0. respectively Feedback inhibition studies showed that ALS is more susceptible to leucine than valine. And $IC_{50}$ value of Cadre, a class of irnidazolinones, is about $1.5\mu{M}$ for ALS.

• 대한바이러스학회지
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• 제28권1호
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• pp.21-30
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• 1998

Synthetic genes encoding the gag p24 and the part of the envelope protein gp41 of the human immunodeficiency virus (HIV-1) were cloned and overexpressed as fusion proteins in Escherichia coli, using an expression vector carrying T7 promoter and the poly-histidine leader, sequence. The overexpressed p24 fusion protein was purified by centrifugation, Ni-affinity chromatography and CM-sepharose chromatography. The overexpressed gp41 fusion protein was purified by centrifugation, $C_4$ chromatography and DEAE-sepharose chromatography. The purified fusion proteins showed a high level of purity and immunoreactivity in SDS-polyacrylamide gel electrophoresis and western blot analysis. These results suggest that this prokaryotic - expression purification method is suitable for obtaining a large amount of the viral antigen which may be useful for screening of antibodies to HIV-1 in human blood samples.

• 한국잠사곤충학회지
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• 제41권2호
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• pp.82-86
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• 1999

Hemolymph proteins and vitellogenin from mulberry longicorn beetle, Apriona germari HOPE, were indentified, and their changed were analyzed during the larval-pupal-adult development and in the newly laid eggs. Thres major hemolymph proteins were observed in the hemolymph during the larval-pupal-adult development and the intensity of their proteins was clearly observed during the pupal stage. From SDS-[olyacrylamide gel electrophoresis analysis, molecular weights of three major hemolymph proteins were approximately 74 kDa, 78kDa and 85kDa. Vitellogenin in A. Germati appeared in the hemolymph of only abult female and is considered to be a product synthersized within 10 days after adult emergence. The molecular weight of vitellogenin was consited of a heavy subunit (165 kDa) and a light subunit (40 kDa).

• BMB Reports
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• 제34권5호
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• pp.415-420
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• 2001

Chloramphenicol acetyltransferase (CAT) was purified to homogeneity from Morganella morganii starting with ammonium sulphate fractionation, followed by separation on DEAE-Sephadex A50, and G-100 Sephadex gel filtration. The enzyme was purified 133.3 fold and showed a final specific activity of 60 units/mg protein with a yield of 37%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it as a heterotetramer that consists of four subunits with close molecular weights (19.5, 19, 18, and 17.5 kDa). The molecular weight of the native enzyme was calculated to be 78 kDa, as determined by gel filtration, which approximated to that of the four subunits (74 kDa). The enzyme showed a maximum activity at pH 7.8 when incubated at $35^{\circ}C$. A Lineweaver-Burk analysis gave a Km of 5.0 uM and Vmax of 153.8 U/ml. The amino acid composition of the purified enzyme was also determined.

• 생명과학회지
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• 제8권4호
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• pp.387-394
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• 1998

A thermophilic bacterium (strain DG0303) producing a thermostable $\alpha$-glucosidase was isolated from manure and identified as Bacillus sp. Strain DG0303 produced high level of $\alpha$-glucosidase compared with other thermophilic Bacillus strains. The cellular protein patterns were also compared with other Bacillus strains by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE). On the basis of 16S rDNA analysis the Bacillus sp. DG0303 was found to be a member of Bacillus rDNA group 5. The optimum temperature for growth was 65$\circ$C and no growth was obtained at 40$\circ$C or 75$\circ$C. The optimum pH for growth was 5.5 to 8.5. $\alpha$-glucosidase activity was produced during growth and most activity was detected in the culture supernatant. The $\alpha$-glucosidase production was constitutive in the absence of carbohydrates. High level of enzyme activity was detected when the culture was grown on medium containing starch. Addition of glucose resulted in the repression of the $\alpha$-glucosidase production. The optimum pH and tempoerature for enzyme activity were pH 5.0 and 65$\circ$C, respectively. When analyzed by zymogram, the culture supernatant showed a single $\alpha$-glucosidase band with a molecular weight of approximately 60,000.