• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1653-1658
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• 2012

We have reported previously that a recombinant Escherichia coli co-expresses aminolevulinic acid (ALA) synthase, an NADP-dependent malic enzyme, and a dicarboxylate transporter-produced heme, an iron-chelated porphyrin, in a succinate-containing complex medium. To develop an industrially plausible process, a chemically defined medium was formulated based on M9 minimal medium. Heme synthesis was enhanced by adding sodium bicarbonate, which strengthened the C4 metabolism required for the precursor metabolite, although a pH change discouraged cell growth. Increasing the medium pH buffering capacity (100mM phosphate buffer) and adding sodium bicarbonate enabled the recombinant E. coli to produce heme at rates 60% greater than those in M9 minimal medium. Adding growth factors (1 mg/l thiamin, 0.01 mg/l biotin, 5 mg/l nicotinic acid, 1 mg/l pantothenic acid, and 1.4 mg/l cobalamin) also induced positive heme production effects at levels twice of heme production in M9-based medium. Porphyrin derivatives and heme were found in the chemically defined medium, and their presence was confirmed by liquid chromatography/mass spectroscopy (LC/MS). The formulated medium allowed for the production of $0.6{\mu}M$ heme, $29{\mu}M$ ALA, $0.07{\mu}M$ coproporphyrin I, $0.21{\mu}M$ coproporphyrin III, and $0.23{\mu}M$ uroporphyrin in a 3 L pH-controlled culture.

• 한국유기농업학회지
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• 제18권2호
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• pp.233-244
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• 2010

본 시험은 육성돈과 비육돈에서 항생제 대체를 위하여 두 가지 첨가제의 가축의 생산성과 사료비를 평가하였다. 본 시험에 사용된 첨가제는 두과식물인 호로파(Trigonella foenum graecum) 추출물(HE)과 aminolevulinic acid(ALA)를 사용하였다. 시험 1은 자돈 128두를 4처리 4반복으로 돈방 당 8두씩 28일간 사양시험을 하였다. 항생제 첨가구(PC)의 일당증체량은 580.6g으로 532.2g의 ALA처리구보다 많이 증체하였다(p<0.05). 한편 일일 사료섭취량과 사료효율은 처리간에 유의적인 차이가 없었다. 자돈의 두당 사료비는 첨가제 처리구인 HE 및 ALA 처리구 모두 항생제 처리구(PC)보다 적었으나, 1.0kg 증체당 사료비는 HE 처리구가 항생제(PC) 처리구보다 높았다. 시험 2는 육성 비육돈 80두를 4처리 4반복으로 돈방당 5두씩 70일간 사양시험을 하였다. 육성 비육돈의 1일 사료섭취량은 HE 처리구가 2,425.8g으로 다른 처리구보다 많이 섭취하였다(p<0.05). 한편 육성 비육돈의 일당증체량과 사료효율은 처리간 유의적인 차이가 없었다. HE 처리구는 두당 사료비와 1.0kg 당 사료비 모두 항생제 처리구보다 많았으나 ALA 처리구는 1.0kg 당 사료비가 항생제 처리구와 같았다. 이상의 시험결과를 종합해 볼 때 항생제를 대체를 위한 첨가제는 가축생산성이 항생제 처리구보다 떨어지지 않았으며, 특히 aminolevulinic acid는 사료비에서도 항생제와 차이가 없어 경쟁력이 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제28권6호
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• pp.796-806
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• 2015

To evaluate the effects of feeding linseed oil or/and sunflower oil mixed with fish oil on milk yield, milk composition and fatty acid (FA) profiles of dairy cows fed a high-concentrate diet, 24 crossbred primiparous lactating dairy cows in early lactation were assigned to a completely randomized design experiment. All cows were fed a high-concentrate basal diet and 0.38 kg dry matter (DM) molasses per day. Treatments were composed of a basal diet without oil supplement (Control), or diets of (DM basis) 3% linseed and fish oils (1:1, w/w, LSO-FO), or 3% sunflower and fish oils (1:1, w/w, SFO-FO), or 3% mixture (1:1:1, w/w) of linseed, sunflower, and fish oils (MIX-O). The animals fed SFO-FO had a 13.12% decrease in total dry matter intake compared with the control diet (p<0.05). No significant change was detected for milk yield; however, the animals fed the diet supplemented with SFO-FO showed a depressed milk fat yield and concentration by 35.42% and 27.20%, respectively, compared to those fed the control diet (p<0.05). Milk c9, t11-conjugated linoleic acid (CLA) proportion increased by 198.11% in the LSO-FO group relative to the control group (p<0.01). Milk C18:3n-3 (ALA) proportion was enhanced by 227.27% supplementing with LSO-FO relative to the control group (p<0.01). The proportions of milk docosahexaenoic acid (DHA) were significantly increased (p<0.01) in the cows fed LSO-FO (0.38%) and MIX-O (0.23%) compared to the control group (0.01%). Dietary inclusion of LSO-FO mainly increased milk c9, t11-CLA, ALA, DHA, and n-3 polyunsaturated fatty acids (PUFA), whereas feeding MIX-O improved preformed FA and unsaturated fatty acids (UFA). While the lowest n-6/n-3 ratio was found in the LSO-FO, the decreased atherogenecity index (AI) and thrombogenicity index (TI) seemed to be more extent in the MIX-O. Therefore, to maximize milk c9, t11-CLA, ALA, DHA, and n-3 PUFA and to minimize milk n-6/n-3 ratio, AI and TI, an ideal supplement would appear to be either LSO-FO or MIX-O.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1521-1526
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• 2007

A maltogenic amylase gene from Lactobacillus gasseri ATCC33323 (LGMA) was expressed in Lactococcus lactis MG1363 using the P170 expression system. The successful production of recombinant LGMA (rLGMA) was confirmed by the catalytic activity of the enzyme in liquid and solid media. The N-terminal amino acid sequencing analysis of the rLGMA showed that it was Met-Gln-Leu-Ala-Ala-Leu-, which was the same as that of genuine protein, meaning the signal peptide was efficiently cleaved during secretion to the extracellular milieu. The optimal reaction temperature and pH of rLGMA ($55^{\circ}C$ and pH 5, respectively) and enzymatic hydrolysis patterns on various substrates (${\beta}$-cyclodextrin, starch, and pullulan) supported that rLGMA was not only efficiently secreted from the Lactococcus lactis MG1363 but was also functionally active. Finally, the branched maltooligosaccharides were effectively produced from liquefied com starch, by using rLGMA secreted from Lactococcus lactis, with a yield of 53.1%.

• Journal of Microbiology
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• 제39권4호
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• pp.305-313
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• 2001

The spr D gene encoding Strptomyces griseus protease D(SGPD); a chymotrypsin-like proteae, was cloned from Strptomyces griseus IFO13350 and sequence. Most of the amino-acid sequence deduced from the nucleotide sequence is idential to that Strptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp 369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The spr D gene was overexpressed in Streptomyce liv-idans TK24 as a heterologous host. Various media with different compositions were also used to max-imize the productivity of SGPD inthe heterologous host. The SGPD productivity was best when the transformant S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-scuccinyl-ala-ala-pro-phe-p-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM medial but it was relatively lower that in R2YE medium and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reacted the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increase in till the 10$^{th}$ day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8days of cultivation. The introduction of the spr D gene into S. lividans TK24 triggered biosyntheis of the pigmented antibiotic , actinorhodin, which implies some protease may paly a very improtant role in secondary-metabolite formation in sStreptomyces.

• KSBB Journal
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• 제18권6호
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• pp.495-499
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• 2003

고온고압수 반응에 의해 생선 내장으로부터 아미노산의 생성거동을 검토하기 위하여 회분식 및 반회분식 반응조작을 행하였으며 그 결과 다음과 같은 결론을 얻을 수 있었다. 25$0^{\circ}C$, 반응시간 60분에서 회분식 반응조작을 통해 얻었던 최적수율 (137nmg/g-dry fish)과 비교하여 상대적으로 고온인 30$0^{\circ}C$, 2분의 반회분식 조작결과 보다 높은 수율 (230mg/g-dry fish)의 개선을 가져올 수 있었다. 또한 회분식 반응조작에서는 Ala, Gly과 같은 비교적 저분자량의 아미노산 생성이 지배적이었으며 반회분식 조작에서는 Ser, Asp과 같은 고분자량의 아미노산 생성이 촉진되었다. 이와 관련하여 반응 생성물의 조성은 각 반응조작 (회분식 및 반회분식)에 의존하는 것을 알 수 있었다 이상의 결과를 토대로 생선 폐기물로부터 아미노산의 생성을 위한 고온고압수 처리공정은 회분식 반응조작에서 얻을 수 있었던 최적온도보다 상대적으로 높은 온도 및 단시간 반응에서 조작되어져야 한다는 것을 알 수 있었으며 특정 아미노산의 수율을 향상시키기 위해서는 보다 정확한 반응조작의 검토가 필요한 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제32권9호
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• pp.1363-1372
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• 2019

Objective: This study was designed to investigate the effect of diet supplementation with rubber seed oil and flaxseed oil on serum fatty acids profile, oxidation stability of serum and milk, and immune function of dairy cows. Methods: Forty-eight mid-lactation Holstein dairy cows were randomly assigned to one of four treatments for 8 wk, including basal diet (CON) or the basal diet supplemented with 4% rubber seed oil (RO), 4% flaxseed oil (FO) or 2% rubber seed oil plus 2% flaxseed oil (RFO) on a dry matter basis. Results: Compared with CON, all the oil groups increased the levels of trans-11 C18:1 (vaccenic acid), cis-9, trans-11 C18:2 (conjugated linoleic acid, CLA) and C18:3 (${\alpha}$-linolenic acid, ALA) in serum. Both the activity of glutathione peroxidase and catalase in serum and milk in oil groups were decreased, which were negatively correlated with the levels of cis-9, trans-11 CLA and ALA. The concentrations of proinflammatory factors (tumor necrosis factor ${\alpha}$ and interferon ${\gamma}$) in serum of oil groups were lower than that from the CON cows. Conclusion: These results indicate that diet supplementation with RO or FO could alter serum fatty acid profile and enhance the immune function of dairy cows. However, the negative effect on milk oxidation stability should be considered when feeding these n-3 polyunsaturated fatty acid-enriched oils in dairy production.

• Asian-Australasian Journal of Animal Sciences
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• 제27권7호
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• pp.1026-1034
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• 2014

This study was conducted to assess the effects of flaxseed oil and dried whitebait as a source of ${\omega}$-3 fatty acids (${\omega}$-3 FA), which could be used to produce eggs enriched with ${\omega}$-3 FA, and of fructo-oligosaccharide (FOS) as a source of prebiotics on performance of hens (commercial Hy-Line Brown laying hens), and FA composition, internal quality, and sensory characteristics of the eggs. Dietary FOS increased egg weight. The amounts of ${\alpha}$-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) in the eggs from the hens fed the flaxseed oil alone or flaxseed oil+dried whitebait diets were higher than those of the control. Hedonic scores for off-flavor, fishy flavor, buttery taste and overall acceptability of the eggs from the hens fed the diet containing flaxseed oil+dried whitebait were lower (p<0.05) than those of the control. Overall acceptability of the eggs from the hens fed the diet containing soybean oil+dried whitebait was lower (p<0.05) than that of the control. However, all the sensory attributes of the eggs from the hens fed the diet containing flaxseed oil, dried whitebait and FOS were not significantly different from those of the control. These results confirmed that flaxseed oil increases the ALA content in the eggs and a combination of flaxseed oil and dried whitebait increases EPA and DHA in the eggs. Of significance was that addition of FOS to the flaxseed oil+dried whitebait diet improves the sensory characteristics of the eggs enriched with ${\omega}$-3 FA.

• Asian-Australasian Journal of Animal Sciences
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• 제28권9호
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• pp.1327-1334
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• 2015

The current study determined the apparent or standardized ileal digestibility of amino acids (AID or SID of AA) in growing pigs fed diets containing three protein feedstuffs with different fiber characteristics at two dietary crude protein (CP) levels. Twenty boars ($Yorkshire{\times}Landrace$) with average initial body weight of $35({\pm}2.6)kg$ were fitted with a simple T-cannula at the distal ileum. These pigs were offered six diets containing soybean meal (SBM), canola meal (CM) or corn distillers dried grains with solubles (corn-DDGS) that were either adequate (19%) or marginal (15%) in CP using a triplicated $6{\times}2$ Youden Square Design. Except for Met, Trp, Cys, and Pro, AID of AA was greater (p<0.05) in the SBM diet compared with the CM diet. Apparent ileal digestibility for Gly and Asp was greater (p<0.05) in the SBM diet compared with the corn-DDGS diet. The AID of Ile, Leu, Phe, Val, Ala, Tyr, and Asp was greater (p<0.05) in the corn-DDGS diet compared with the CM diet. Standardized ileal digestibility of AA was greater (p<0.05) in the SBM diet compared with the CM diet for all AA except Trp and Pro. The SID of Ile, Leu, Val, Ala, Tyr, and Asp was greater (p<0.05) in the corn-DDGS diet compared with the CM diet. It was concluded that protein feedstuff affects ileal AA digestibility and is closely related to dietary fiber characteristics, and a 4-percentage unit reduction in dietary CP had no effect on ileal AA digestibility in growing pigs.