Candida magnoliae HH-01에 의한 과당으로부터 만니톨로의 생전환

다국어 초록 (Multilingual Abstract)

The purpose of this thesis was to develop the optimal fermentation processes for the production of mannitol. For this purpose, isolation of producer strains, optimization of fermentation conditions such as carbon/nitrogen source, pH, temperature etc, and purification and characterization of key enzyme involved in mannitol biosynthesis were investigated.
A novel microorganism which is able to produce mannitol when grown in the presence of fructose and glucose as a carbon source was isolated from the fermented product using various vegetables. The isolated strain was identified as Candida magnoliae based on the identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.
In this study, NADPH-dependent mannitol dehydrogenase from C. magnoliae HH-01 was purified to homogeneity by ion-exchange chromatography. The relative molecular masses of C. magnoliae mannitol dehydrogenase, as determined by sodium dedecyl sulfate-polyacrylamide gel electrophoresis was approximately 35 kDa. The enzyme catalyzed reduction of fructose into mannitol. The pH and temperature optima for fructose reduction was 7.0 and 35??, respectively. C. magnoliae mannitol dehydrogenase showed the highest activity with fructose as the substrate and used only NADPH. The enzyme activity was inhibited by CuCl2 and sulfhydryl compounds but not by EDTA.
The mdh gene encodes mannitol dehydrogenase, an enzyme to convert fructose to mannitol. The oligonucleotides deduced from the peptide sequences and random primer were used to prepare the putative mdh gene from the chromosomal DNA of C. magnoliae by PCR. The gene encoding the enzyme was cloned and sequenced. The results demonstrated that a mdh gene consists of 852 bp and encodes 283 amino acids. Based on the cofactor binding site (GXXXGIG), structural stabilizing motif (DXXXNNAG), the active center (YXASK), catalysis enhancing site (PG), and H-bonding to carboxamide of nicotinamide ring (T), mannitol dehydrogenase has been placed with the NADPH-dependent short chain dehydrogenases/reductases (SDRs). The deduced amino acid sequences of C. magnoliae mannitol dehydrogenase showed a significant homology with SDRs from various sources, indicating that the C. magnoliae mannitol dehydrogenase is an NADPH-dependent tetrameric SDR. After fusion of six histidine codons to the 3' end of mdh gene and expression in E. coli, the active mannitol dehydrogenase from the culture broth of the recombinant E. coli was could be purified in a two-step procedure by affinity chromatography using a Ni+-NTA matrix column. For the study of the mdh gene expression, the putative promoter was isolated. GFP, a reporter protein, was expressed under the control of putative 158 bp fragment in C. magnoliae.
To enhance the production of mannitol, cultivation conditions and medium composition were optimized. The most efficient substrate for mannitol production by C. magnoliae HH-01 was fructose. Glucose and sucrose also could also be converted into mannitol but with lower conversion yields. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration. The optimal pH and temperature for mannitol production were 4.5 - 5.0 and 33??, respectively. Under optimum conditions, a final mannitol production of 323 g․l-1 was obtained 333 g fructose․l-1 after 164 h, corresponding to an 1.97 g․l-1․h-1 and 97 % yield.

국문 초록 (Abstract)

본 논문의 목적은 만니톨 생산을 위한 최적의 발효공정을 개발하는데 있다. 이 를 위해서 만니톨 생산균주의 분리 및 동정, 발효조건(탄소원, 질소원, pH, 온도) 의 최적화, 그리고 만니톨 생산에 관여하는 효소에 대한 정제 및 특성을 조사하 고자 하였다.
만니톨을 생산하는 새로운 미생물을 선별하기 위해서 다양한 장소에서 시료를 채취하였으며 이들을 과당과 포도당이 과량 함유된 선별배지에서 배양하였다. 그 결과 만니톨을 과량 생산하는 미생물을 선별하였으며 선별된 균주을 동정하기 위해서 26S rDNA의 D1/D2 domain의 염기서열을 근거로 하여 Candida magnoliae 임을 밝혀 내었다. 또한 선별한 균주와 C. magnoliae reference strain 의 탄소원 비교를 통해서 같은 종임을 밝혀 내었다.
본 연구에서 C. magnoliae로부터 NADPH-dependent mannitol dehydrogenase 을 이온교환 크로마토그래피을 이용하여 분리 정제를 하였다. Mannitol dehydrogenase의 분자량을 알아보기 위해서 SDS-PAGE을 수행하였으며 그 결 과 약 35 kDa의 크기를 가짐을 확인하였다. 이 효소는 과당에서 만니톨로 환원 시키는 반응을 촉매한다. 또한 과당 환원에 대한 최적 pH와 온도는 각각 7.0과 35℃였다. C. magnoliae에서 만니톨의 생산에 관여하는 효소의 기질과 조효소에 대한 특성을 알아보았으며 기질로 과당을 조효소로 NADPH을 사용하였을때 가 장 높은 활성을 보였다. 그리고 CuCl2와 sulfhydryl compounds에 의해 효소 활 성이 저해되었으나 EDTA는 전혀 영향을 주지 않았다.
Mannitol dehydrogenase을 coding하는 mdh 유전자를 cloning하였다. Putative mdh 유전자를 얻기 위해서 C. magnoliae chromosomal DNA와 아미노산 서열 분석을 통해서 얻은 deduced oligonucleotides와 random primer을 사용하여 PCR 을 수행하였다. 그 결과 852 bp의 mannitol dehydrogenase gene을 얻었으며 유 전자 분석을 통해서 mannitol dehydrogenase가 283 개의 아미노산으로 구성되어 있음을 확인하였다. 또한 deduced amino acid 분석을 통해서 cofactor binding site (GXXXGIG), structural stabilizing motif (DXXXNNAG), the active center (YXASK), catalysis enhancing site (PG), 그리고 H-bonding to carboxamide of nicotinamide ring (T)을 확인하였으며 이와 같은 결과를 토대로 C. magnoliae mannitol dehydrogenase는 NADPH-dependent Short chain dehydrogenases /reductases(SDR)임을 확인하였다. C. magnoliae의 mannitol dehydrogenase는 다양한 다른 미생물의 SDR과 많은 유사성을 보였으며 NADPH-dependent tetrameric SDR임을 확인하였다. mdh 유전자의 3‘ 끝부분에 6개의 히스티딘 codon을 결합시킨 다음 대장균에서 발현하였으며 Ni+-NTA agarose에서 분리정 제를 수행하였다. mdh 유전자의 발현에 대한 연구를 위해서 putative promoter를 분리하였다. C. magnoliae에서 158bp의 putative promoter region에 의해서 GFP protein이 발현함을 확인하였다.
만니톨 생산의 최적화를 위해서 배양조건과 배양배지을 최적화 하였다. C. magnoliae에서 만니톨 생산에 최적의 탄소원은 과당이었다. 또한 포도당과 설탕 에서는 만니톨로 전환은 되지만 낮은 생산성을 보였다. Fed-batch fermentation 에서 포도당이 고갈 되었을때 과당에서 만니톨로의 전환이 감소하게 된다. 이때 다시 포도당을 첨가하면 과당에서 만니톨로의 전환이 다시 일어나게 된다. 이는 포도당이 만니톨의 생산에서 조효소인 NADPH의 regeneration에 중요한 역할을 담당하고 있다는 것을 내포하고 있다. 발효조건(pH, 온도등) 최적화를 수행하였 으며 pH 5.0, 33℃에서 배양시간 164시간동안 333 g l-1의 과당에서 323 g l-1 의 만니톨을 생산하며 1.97 g l-1 h-1의 productivity와 97 %의 yield로 만니톨을 생 산한다.

목차 (Table of Contents)

• Chaper I. Literature review = 1
• 1. Introduction = 2
• 1.1 Sugar alcohols = 2
• 1.2 Distribution and phycochemical properties of mannitol = 3
• 1.3 Microbial production of mannitol = 4
• 1.3.1 Bacteria = 4
• 1.3.2 Yeast = 6
• 1.3.3 Fungi = 7
• 1.4 Enzymatic conversion of fructose into mannitol = 8
• 1.4.1 Conversion by Mannitol dehydrogenase = 8
• 1.4.2 Conversion by Mannitol 1-phosphate dehydrogenase = 9
• 1.5 Recovery of mannitol = 10
• 1.6 Reference = 13
• Chaper II. Screening of a New Mannitol Producing Strain = 27
• Abstract = 28
• 1. Introduction = 29
• 2. Materials and Methods = 30
• 2.1 Microorganism and culture conditions = 30
• 2.2 Analytical methods = 30
• 2.3 Sequence determination = 30
• 3. Results = 32
• 3.1 Isolation of mannitol-producing strain = 32
• 4. Reference = 34
• Chpater III. Purification and Characterization of mannitol dehydrogenasefrom Candida magnoliae HH-01 = 41
• Abstract = 42
• 1. Introduction = 43
• 2. Materials and Methods = 44
• 2.1 Microorganism and culture conditions = 44
• 2.2 Analytical methods = 45
• 2.3 Assay for mannitol dehydrogenase activity = 45
• 2.4 Purification of mannitol dehydrogenase from C. magnoliae = 46
• 2.5 PAGE and activity staining = 47
• 2.6 Peptide sequence determination = 48
• 2.7 Amino acid composition = 49
• 3. Results = 49
• 3.1 Purification of mannitol dehydrogenase from C. magnoliae = 49
• 3.2 Substrate and coenzyme specificity of mannitol dehydrogenase = 50
• 3.3 Peptide sequence and amino acid composition of mannitol dehydrogenase = 50
• 3.4 Effect of temperature on stability and activity of mannitol dehydrogenase = 51
• 3.5 Effect of pH on activity of mannitol dehydrogenase = 51
• 3.6 Effect of metal ions and various compounds = 51
• 4. Reference = 55
• Chapter IV. Molecular cloning, Sequencing and Characterization of mannitol dehydrogenase gene in C. magnoliae HH-01 = 70
• Abstract = 71
• 1. Introduction = 72
• 2. Materials and Methods = 73
• 2.1 Bacterial strain and plasmid = 73
• 2.2 Culture and transformation conditions = 73
• 2.2.1 E. coli = 73
• 2.2.2 C. magnoliae = 74
• 2.3 Purification of mannitol dehydrogenase = 75
• 2.4 PAGE = 75
• 2.5 Cloning of mdh promoter region = 76
• 2.6 Fluorescence measurement of expressed GFP = 76
• 2.7 PCR amplification = 76
• 2.8 Construction and screening of a genomic DNA library = 77
• 2.9 Cloning of mdh gene in E. coli expression vector = 77
• 3. Results = 78
• 3.1 Cloning and sequence analysis of mannitol dehydrogenase gene = 78
• 3.2 Sequence of the mannitol dehydrogenase from C. magnoliae as derived from the DNA sequence = 78
• 3.3 Construction of an expression vector = 79
• 3.4 Overexpression and purification of mannitol dehydrogenase in E. coli = 79
• 3.5 Molecular analysis of C. magnoliae promoter = 80
• 4. Reference = 81
• Chapter V. Optimization of fermentation process for mannitol production by C. magnoliae HH-01 = 97
• Abstract = 98
• 1. Introduction = 99
• 2. Materials and Methods = 100
• 2.1 Microorganism and culture conditions = 100
• 2.2 Analytic methods = 100
• 3. Results = 101
• 3.1 Effect of carbon and nitrogen source = 101
• 3.1.1 Carbon source = 101
• 3.1.2 Nitrogen source = 101
• 3.2 Optimization of fermentation conditions = 102
• 3.2.1 Effect of glucose limitation on the production of mannitol = 102
• 3.2.2 Effect of pH on the production of mannitol = 103
• 3.2.3 Effect of temperature on the production of mannitol = 103
• 4. Reference = 106
• 국문초록 = 121
• 감사의글 = 123
• Appendix = 124