• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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• pp.723-731
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• 2008

Immunomodulatory feed additives might offer alternatives to antimicrobial growth promoters in pig production. This experiment was designed to determine the effects of dietary galacto-mannan-oligosaccharide (GMOS) and chitosan oligosaccharide (COS) supplementation on the immune response in early-weaned piglets. Forty 15-day-old piglets (Duroc$\times$Landrace$\times$Yorkshire) with an average live body weight of $5.6{\pm}0.51kg$ were weaned and randomly assigned to 4 treatment groups that were fed maize-soybean meal diets containing either basal, 110 mg/kg of lincomycin, 250 mg/kg of COS or 0.2% GMOS, respectively, over a 2-week period. Another six piglets of the same age were sacrificed on the same day at the beginning of the study for sampling, in order to obtain baseline values. Interleukin (IL)-1${\beta}$gene expression in peripheral blood monocytes, jejunal mucosa and lymph nodes, as well as serum levels of IL-1${\beta}$ IL-2 and IL-6, IgA, IgG, and IgM, were evaluated for 5 pigs from each group at 15 and 28 days of age. The results indicate that weaning stress resulted in decreases in serum antibody and cytokine levels. Dietary supplementation with GMOS or COS enhanced (p<0.05) IL-1${\beta}$gene expression in jejunal mucosa and lymph nodes, as well as serum levels of IL-1${\beta}$ IL-2, IL-6, IgA, IgG and IgM compared to supplementation with lincomycin. These findings suggest that GMOS or COS may enhance the cell-mediated immune response in early-weaned piglets by modulating the production of cytokines and antibodies, which shows that GMOS or COS have different effects than the antibiotic on animal growth and health.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.347-355
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• 2016

An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50℃; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50℃ than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of V_max and k_cat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

• 한국미생물·생명공학회지
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• 제46권1호
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• pp.45-50
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• 2018

The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.

• 미생물학회지
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• 제42권2호
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• pp.142-148
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• 2006

본 연구는 다양한 생리활성을 가지고 있는 chitosan oligosaccharides를 효소적 방법으로 생산하기 위한 기초 연구로서, 전통발효식품인 메주에서 chitosan 분해능이 우수한 균주를 분리하였다. 분리한 균주를 형태학적, 생화학적 및 분자생물학적 방법을 이용하여 동정한 결과, Bacillus amyloliquefaciene MJ-1으로 명명하였다. B. amyloliquefaciene MJ-1으로 부터 chitosanase 유전자를 포함하는 1,049 bp DNA 단편을 클로닝하였으며, chitosanase 유전자는 825 염기로서 274 개의 아미노산으로 구성되어 있었고, 예상 분자랸은 30.9 kDa이었다. 클로링한 chitosanase의 homology search 결과, glucoside hydrolase family 46에 속하는 chitosanase로 추정되었다. B. amyloliquefaciene MJ-1 chitosanase 유전자를 E. coli BLR (DE3)에 도입하였으며, 1 mM의 IPTG로 chitosanase 과잉 발현을 유도하고 정제한 후, pH및 온도에 대한 특성을 조사하였다. 효소 활성의 최적 온도는 $60^{\circ}C$이었으며, $80^{\circ}C$에서도 75%의 활성을 나타내었으므로 내열성을 가진 효소로 추정되었다. 한편, 최적 pH는 5.0 이었으며, pH $5{\sim}7$사이에서 80% 이상의 높은 활성을 유지하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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• pp.556-562
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• 2007

A total of 126 crossbred weanling pigs (average body weight of $6.3{\pm}0.3$ kg) were used to investigate the effect of chito-oligosaccharide (COS) on growth performance, nutrient digestibility, pH of gastro-intestinal tract (GI), intestinal and fecal microflora of young piglets. Pigs were allocated to three dietary treatments based on body weight and gender in a single factorial arrangement. Treatments were control (No COS), T1 (0.2% COS during starter (6-13 kg) and 0.1% COS during grower (13-30 kg) phases, and T2 (0.4% COS during starter (6-13 kg) and 0.3% COS during grower (13-30 kg) phases, respectively. Each treatment had 3 replicates and 14 pigs were raised in each pen. COS is a low molecular weight water-soluble chitosan that can be obtained from chitin of the crab shell after deacetylation with concentrated sodium hydroxide at high temperature and then further decomposition by chitosanase enzyme in the presence of ascorbic acid. For the starter and grower periods, there were no significant differences (p>0.05) in average daily gain (ADG) and feed to gain ratio among treatments. However, during the overall period (6-30 kg), T2 showed better (p<0.05) feed to gain ratio than other treatments. A digestibility study was conducted at the end of grower phase which showed improvement (p<0.05) in DM and crude fat digestibility in T2 over the control. At 25 kg body weight, 6 pigs per treatment (2 per replicate) were sacrificed to determine the effect of diets on pH and microbial count at different sections of the GI tract. The pH of the cecal contents in pigs fed 0.1% COS was higher (p<0.05) than in the other treatments. Total anaerobic bacterial number increased from cecum to rectum in all treatments. The weekly total bacterial counts showed higher (p<0.05) in feces of pigs fed COS than that of untreated pigs at the $8^{th}$ week. The number of fecal E. coli in untreated pigs at $4^{th}$ wk was 7.35 log CFU/g compared to 6.71 and 6.54 log CFU/g in 0.1 and 0.3% COS-treated pigs, respectively. Similarly, at $8^{th}$ wk, fecal clostridium spp. were lower in pigs fed 0.3% COS (5.43 log CFU/g) than in untreated pigs (6.26 log CFU/g). In conclusion, these results indicated that chito-oligosaccharide could improve feed efficiency in young pigs and inhibited the growth of harmful bacteria.

• KSBB Journal
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• 제16권4호
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• pp.321-326
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• 2001

막의 특성을 이용하여 기질과 효소가 공존하는 캡슐을 제조하여 이 캡슐의 장점인 membrane의 pore size를 조절하면 올리고당을 분자량별로 유출시키는 것이 가능해짐으로 기질과 효소의 유출을 조절하여 생리활성이 높은 올리거당을 얻고, 생산된 올리고당의 단순한 분리.정제를 위하여, 그리고 효소의 재 이용 측면에서 encapsulation technology를 이용하여 올리고당을 생산하는 연구를 하였다. 제조된 capsule의 막 두께와 직경은 Olympus IX50 microscope를 이용하여 측정한 결과 막의 두께는 10 $\mu$m이었으며, capsule의 크기는 대략 3.0mm, $N_2$ gas를 이용할 capsule은 대fir 1.5 mm였다. 본 실험에 사용된 기질의 용액, 효소액, 반응액, alginate 용액을 pH 5.0으로 제조하여 사용하였기 때문에 막이 보다 견고하게 형성 할 수 있었으며, 제조된 capsule를 반응 용액 즉 2% acetic acid의 pH를 CaCO$_3$로 조절한 용액에 넣었기 때문에 막이 단단하게 형성되고 유지되는 것으로 판단된다. 기질의 사용에 있어서 작은 분자량을 갖는 기질은 효소와 분해 반응에 있어서 가수분해가 빨리 일어나기 때문에 보통 capsule 밖으로 유출된 올리고당의 분자량은 평균 M.W. 1,000이었다. 그러나 분자량이 큰 기질을 사용하였을 경우 평균 M.W. 1,000-3,500이었다. 막을 이루는 alginate의 점도에 의한 차이는 없었으며, 교반 속도에 의한 기질과 효소의 유출, 그리고 분해 반응에 따른 가수분해 산물인 올리고당의 유출에 있어 alginate 농도 0.5%, 교반 속도는 40 rpm이 가장 적당하였다. (12). Capsule에서 분해 반응 후 분해 산물인 올리고당의 이동을 빨리 하기 위하여 작은 size의 capsule를 제조하여 실험한 결과 3.0 mm보다 capsule 내부의 분자량 변화가 더 빨랐으며, capsule 밖으로 유출된 올리고당의 분자량 분석 결과 M.W. 3000-6000의 올리고당이 생성되었다. 반응 시간에 따른 capsule 내부의 잔류 효소 활성을 조사한 결과, 반응 8시간째에도 약 80% 이상의 효소활성을 나타내었다. Batch-type과 비교하여 capsule의 경우에는 반응이 끝난 후 capsule를 회수하여 효소를 재 사용할 수 있을 것으로 사료된다.