초록 ▼

○ 연구결과
Proteus mirabilis 유래의 두 amino acid deaminase 유전자 pma와 pm1의 대장균 발현 시스템이 구축되었고 활성형의 Pma, Pm1 효소가 대량 생산될 수 있도록 대량생산 시스템이 확립되어 PLA가 96% 이상 회수율을 보였으며, 대량생산된 항균성 유기산 phenyllactic acid가 적량 함유된 양돈, 양계용시제품을 각각 제조하였음. 사양시험 결과 이유자돈에 있어 소화율 개선과 그에 따른 면역력 향상 및 장내 유익균의 증가와 유해균의 감소로 인해 이유자돈 시기에 나타날 수 있는

○ 연구결과
Proteus mirabilis 유래의 두 amino acid deaminase 유전자 pma와 pm1의 대장균 발현 시스템이 구축되었고 활성형의 Pma, Pm1 효소가 대량 생산될 수 있도록 대량생산 시스템이 확립되어 PLA가 96% 이상 회수율을 보였으며, 대량생산된 항균성 유기산 phenyllactic acid가 적량 함유된 양돈, 양계용시제품을 각각 제조하였음. 사양시험 결과 이유자돈에 있어 소화율 개선과 그에 따른 면역력 향상 및 장내 유익균의 증가와 유해균의 감소로 인해 이유자돈 시기에 나타날 수 있는 성장률 감소를 예방할 수 있고 육성돈에 있어 0.2%의 유기산 첨가 시 소화율 개선에 의해 사료효율이 향상되었으며, 0.3%의 유기산 첨가 시 면역력 증진과 장내 유해균이 감소하는 효과를 보여 성장을 목적으로 첨가할 경우 0.2%의 유기산을, 성장률 감소현상이 나타날 경우 0.3%의 유기산을 첨가함으로써 다목적 활용이 가능하리라 사료됨. 산란계에 있어서 난각 강도, 난중, 난황색, 호우유닛의 개선에 의한 계란품질의 향상 및 면역력 증가를 보였으며 육계에 있어서는 유기산 첨가에 의한 사료요구율 향상과 면역력 증진 및 육질 개선이라는 효과로 인한 농가의 생산성 증대를 가져올 것으로 기대됨.

Abstract ▼

Ⅳ Results of research development
1. Construction and characterization of AAD expression vecter system
The E. coli expression system of pma and pm1 which are two amino acid deaminase genes from Proteus mirabilis was constructed and the active-formed Pma and Pm1 enzyme were produced in a large

Ⅳ Results of research development
1. Construction and characterization of AAD expression vecter system
The E. coli expression system of pma and pm1 which are two amino acid deaminase genes from Proteus mirabilis was constructed and the active-formed Pma and Pm1 enzyme were produced in a large quantity.
The active-formed Pma and Pm1 enzyme in E. coli are confirmed as membrane-bounded form and the membrane structure may play a important role to activate Pma and Pm1 enzyme. In addition, the membrane structure of Pma and Pm1 inhibits the purification of enzyme proteins.
Pma had high enzyme activity with aromatic amino acids such as phenylalanine, while Pm1 had high enzyme activity with non-aromatic amino acids such as histidine. Km and Vmax of Pma against phenylalanine is 8.66 mM and 0.93 μmol/min mg mg and Km and Vmax of Pm1 against histidine is 4.96 mM and 1.09 μmol/min mg.
It was confirmed that The reaction of Pma cause generation of phenylpyrivic acid (PPA) which is a precusor of phenyllactic acid (PLA) from phenylalanine. This result showed that this amino acid deaminase would be efficiently applied to the development of PLA production process with a Lactobacillus containing the enzyme activity of phenylpyruvate dehydrogenase which can convert PPA to PLA.
The system was also used Bacillus megaterium which is a more safe microbe and constructed for mass production of Pma enzyme. Pma enzyme was produced well from the constructed system, but unfortunately the enzyme was inactive form. It might arise from physiological difference between gram negative and positive bacteria
2. Mass production of antimicrobial organic acid
For mass production of antibacterial organic acids, Pma which is an excellent transformant having strong enzyme activity was used and the mass production system such as optimum of enzyme mass production, bioconversion process, and downstream process, etc. was established. The system was successfully set up for 500 L of mass production and finally scaling up to 2 M/T volume. The optimal condition for enzyme mass production was that TB media was best for bacterial growth and the highest activity of enzyme was at 0.1% lactose.
The enzyme activity was 42.7 unit/ml at 6 hr of expression induction under the optimal condition which is 2% phenylalanine. In downstream process, the CaCl2 selected among various mono, di-, trivalent metal ions was used to induce precipitation for maximum recovery of antibacterial organic acid. After drying of the precipitate using heated-air drying, the recovery rate was calculated to 96% excellently. Based on these data, the prototypes of feed additives containing phenyllactic acid obtained from mass production were manufactured for swine and poultry.
3. Effect of dietary phenyllactic acid(PLA) supplementation in swine.
1) In nursery pigs
90 nursery pigs were used in experiment. 3 dietary treatments were:
1) negative control (NC),
2) positive control (PC), NC + antibiotics, and
3) PLA, NC + 0.5% PLA. In Exp. 2, dietary treatments were: 1) control diet (CON), 2) PLA-0.1, CON + 0.1% PLA, 3) PLA-0.2, CON + 0.2% PLA, and 4) PLA-0.3, CON + 0.3% PLA. Pigs fed the PC and PLA diets had greater ADFI during the overall period (P<0.05), and tended to have greater ADG and G:F from d 7 to d 21 (P<0.10) than those fed the NC diet. The ATTD of DM was greatest in pigs fed the PLA diet at d20 an d41, and N digestibility at d20 was higher in pigs fed the PLA diet(P<0.05) than those fed the NC diet. The numbers of white blood cell(WBC) and lymphocyte concentrations at d 42 were increased (P<0.05; P < 0.01) by the inclusion of antibiotics and PLA in the diet.
2) In growing pigs
96 growing pigs were used in experiment. Dietary treatments were: 1) control diet (CON), 2) PLA-0.1, CON + 0.1% PLA, 3) PLA-0.2, CON + 0.2% PLA, and 4) PLA-0.3, CON + 0.3% PLA. G:F tended to increase when PLA was added (quadratic, P<0.10). The ATTD of DM did not differ among treatments, but there was a tendency (quadratic, P<0.10) for N digestibility to increaseas PLA levels increased. The lymphocyte percentage at d 42 increased linearly as dietary PLA increased (P<0.05).
Additionally, the WBC counts at d 42 tended to increaseas PLA levels increased (P<0.10). In both experiments, there was no effect of treatment on the fecal pH or presence of lactobacillus, but the number of E.coli in feces at d 41 decreased in response to the addition of PLA (P<0.001).
4. Effect of dietary phenyllactic acid(PLA) supplementation in poultry.
1) In laying hens
240 ISA brown laying hens were used in experiment. Dietary treatments were: 1) CON (basal diet), 2) PLA-0.1 (CON + 0.1% phenyllactic acid), 3) PLA-0.2 (CON + 0.2% phenyllactic acid) 및 4) PLA-0.3 (CON + 0.3% phenyllactic acid). Whereas supplementing the diet with PLA did not affect average daily feed intake and feed efficiency, it linearly improved egg production rate, eggshell breaking strength and Haugh unit. The egg weight, eggshell thickness and egg yolk color were not significantly altered by supplementing the diet with PLA. White blood cell, red blood cell, total protein and albumin concentrations were higher in PLA1 and CON treatment groups (P<0.05), while the groups fed the PLA-0.2 and PLA-0.3 diet had the greater (P<0.05) lymphocyte concentration.
2) In broilers
500 2-d-old male Arbor Acres broiler chicks were used in experiment. Dietary treatments were: 1) CON (basal diet), 2) ANT (basal diet + 44 mg/kg avilamycin), 3) PLA0.2 (basal diet + 0.2% PLA), 4) PLA0.4 (basal diet + 0.4% PLA), 5) PLA0.2 (basal diet + 0.2% PLA + 44 mg/kg avilamycin). Weight gain (BWG) were linearly decreased (P<0.05) by the effect of PLA during d 0 to 7 d, while it was increased (P<0.05) by the addition of antibiotics during d 21 to 35. The effect of PLA or antibiotics alone and interaction were observed to reduce (P<0.05) feed intake during the whole experiment except d 8 to 21. During d 0 to 7, the FCR was higher (P<0.05) in antibiotics group than other treatments. While PLA were not observed to affect the FCR during the first 21 d and overall period, it linearly decreased (P<0.05) feed conversion during d 21 to 35. Supplementation diets with PLA and antibiotics resulted in lower E.coli population in small intestine (P<0.01). Moreover, the effect of antibiotics or PLA alone or together was show to reduce the E.coli population in large intestine (P<0.05), and a linearly effect were evident as PLA levels increased (P<0.05). The relative weights of gizzard, liver, spleen, bursa of fabricius, breast, and abdominal fat were not affected by any dietary supplementations. Neither PLA nor antibiotics supplementation had effect on the red blood cell (RBC) and immuoglobulin (IgG) concentrations. However, the white blood cell (WBC) counts and lymphocyte percentage were higher in PLA treatment than non PLA supplementation treatment (P<0.05). The means of L*, a* and pH value did not differ among treatment groups. However, the b* value decreased (linear, P<0.05) as the levels of PLA in the diet increased from 0 to 0.4%.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 12
• CONTENTS ... 22
• 목차 ... 26
• 제1장 연구개발과제의개요 ... 29
• 제1절 연구개발의 중요성 ... 29
• 1. 경제적 중요성 ... 29
• 2. 산업적 중요성 ... 30
• 제2절 연구개발의 필요성 ... 32
• 제3절 연구 개발의 내용 및 범위 ... 36
• 1. 연구개발의 목표 ... 36
• 2. 연차별 연구개발의 내용 및 범위 ... 37
• 제2장 국내외 기술개발 현황 ... 40
• 제1절 국내 현황 ... 40
• 제2절 국외 현황 ... 43
• 제3장 연구개발수행 내용 및 결과 ... 47
• 제1절 탈아미노화효소 발현시스템구축 및 특성 연구 ... 47
• 1. 연구목적 ... 47
• 2. AAD 유전자의 클로닝과 발현시스템의 구축 ... 49
• 3. AAD 유전자의 발현 특성 분석 ... 55
• 4. Pma, Pm1 효소의 활성 분석 ... 64
• 5. Phenylpyruvic acid 생산 ... 68
• 6. Bacillus megaterium을 이용한 pma 발현 시스템의 개발 ... 70
• 7. 결론 및 토론 ... 74
• 제2절 항균성 유기산 함유 사료첨가제 개발 ... 75
• 1. 연구목적 ... 75
• 2. 효소 대량 생산 최적화 ... 76
• 3. 생물전환 공정 확립 ... 81
• 4. Downstream 공정최적화 ... 84
• 5. 생물전환을 통한 항균성 유기산 PLA 대량생산 및 시제품 제조 ... 87
• 6. 결론 및 토론 ... 92
• 제3절 항생제 대체를 위한 유기산 첨가에 따른 생산성 조사 ... 93
• 1. 최종생산물 안전성 평가 ... 93
• 2. 최종생산물 기능성 평가 ... 95
• 3. 적용제품 기초 자료 완성 ... 98
• 4. 양돈시제품성능시험 ... 98
• 5. 양계시제품성능시험 ... 101
• 6. 양돈사료 첨가 급여 후 첨가 수준 결정을 위한 사양시험으로 생산성 향상 여부 평가 ... 105
• 7. 양계사료 첨가 급여 후 첨가 수준 결정을 위한 사양시험으로 생산성 향상 여부 평가 ... 117
• 제4장 목표달성도 및 관련분야에의 기여도 ... 129
• 제1절 목표달성도 ... 129
• 제 1 절 관련분야의 기여도 ... 135
• 제5장 연구개발 성과 및 성과활용 계획 ... 136
• 제1절 실용화.산업화 계획 ... 136
• 제2절 교육 지도 홍보 등 기술확산 계획 ... 140
• 제3절 특허, 논문 등 지식재산권 확보계획 ... 141
• 제4절 추가연구, 타연구에 활용 계획 ... 142
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 144
• 제7장 참고문헌 ... 148
• 끝페이지 ... 158