초록

○ 연구결과
- 유당 분해후 생성된 갈락토스로부터 타가토스 생산: 효소활성율 98.5%, 생물반응기에서 전환수율 53%, 생산성 32 g/L-h, 40일 작동, 정제수율 87.5%
- 유당 가수분해 조건최적화
- 유전자 진화를 통한 타가토스 생산 효소 개량
- 고정화 효소 및 균체를 이용한 생물반응기에서 타가토스 생산
- 유당으로부터 시작하여 타가토스까지의 생물학적 전환공정 확립
- 타가토스 시제품 제조: 70 g 제조

Abstract ▼

1. Lactose hydrolysis: Kluyveromyces lactis β-galactosidase gene was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the β-galactosidase

1. Lactose hydrolysis: Kluyveromyces lactis β-galactosidase gene was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the β-galactosidase was the major fraction but had a lower activity than those of multimeric forms. The purified enzyme required Mn2+ for activity. The activity was optimal at 40℃ and the optimum pH value was 7.0.
A putative β-galactosidase gene of Thermotoga maritima was expressed in E. coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1,100 amino-acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperature for β-galactosidase activity was 80℃ and the optimum pH value was 6.5. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90℃ were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for β-galactosidase activity.
Among K. lactis β-galactosidase, T. martima β-galactosidase, lactozym (β-galactosidase purchased from Novo Co.), Lactozym had the highest activity for lactose hydrolysis at the same concentration of protein. Therefore, we used lactozym in lactose hydrolysis.
2. Strain isolation and DNA cloning for D-tagatose production: A strain, producing bacterial thermostable L-arabinose isomerase, was isolated from Korean soil samples obtained from compost under high temperature circumstances. This strain was identified as Geobacillus thermodenitrificans based on the 16S rRNA analysis, and biological and biochemical characteristics. The araA gene, encoding L-arabinose isomerase (AI), from G. thermodenitrificans was cloned basis on the L-arabinose isomerase gene of G. stearothermophilus and expressed in E. coli.
3. Increase in D-tagatose production by site-directed mutagenesis: L-Arabinose isomerase from G. stearothermophilus has been genetically evolved to increase the reaction rate toward D-galactose. The single point mutations influencing the activity were investigated based on the sequence of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val⁴⁰⁸ and Asn⁴⁷⁵ were determined as the high influencing mutation points for D-galactose isomerization activity. Random mutation was introduced into the sites Val⁴⁰⁸ and Asn⁴⁷⁵ (X408V and X475N) and candidates were screened based on the non-optimal pH condition. Among the random mutations at the site 408 and 475, mutations of Q408V and R408Vwere selected. The optimal pH of the both mutations Q408V and R408V were shifted to pH 7.5. At the shifted optimal pH 7.5, the D-galactose isomerization activity of Q408V and R408V were 60% and 30% higher than that of wild type at pH 8.5.
Among single-site mutations of l-arabinose isomerase derived from G. thermodenitrificans, the W164G and N475K mutants showed the lowest and highest activity,respectively, for D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at site 164 and the size of site 475 were important for D-tagatose production. Among double-site mutations, the C450S-N475K mutant exhibited the highest d-tagatose production. After 300 min at 65C, the d-tagatose yield from d-galactose was 46% for the wild-type, 55% for the N475K mutant, and 58% for the C450S-N475K mutant.
4. Characterization of wild and mutant L-arabinose isomerase: A mutated gene was obtained by an error-prone polymerase chain reaction using the l-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in E. coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322→Val, Ser393→Thr, and Val408→Ala), compared to the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerisation activity at pH 8.0, 65℃, and 1.0 mM Co²⁺, while the wild-type enzyme had a maximum activity at pH 8.0, 60℃, and 1.0 mM Mn²⁺. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerisation activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose.
The araA gene, encoding L-arabinose isomerase (AI), from G. thermodenitrificans was cloned and expressed in E. coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70℃ and pH 8.5 in the presence of Mn²⁺. The enzyme was stable at temperatures below 60℃ and within the pH range 7.5-8.0. D-Galactose and L-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5 mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for D-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM^-1 min^-1 for L-arabinose and 0.5 mM^-1 min^-1 for D-galactose. Mn²⁺ was a competitive activator and increased the thermal stability of the AI. The d-tagatose yield produced by AI from D-galactose was 46% without the addition of Mn²⁺ and 48% with Mn²⁺ after 300 min at 65℃
5. Tagatose prodcution by immobilized cells: Using immobilized recombinant E. coli cells containing G. stearothermophilus larabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70℃ and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60℃ and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h^-1, respectively. Under the optimum conditions, the immobilized cell reactor with Mn²⁺ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L-h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.
6. Tagatose prodcution by immobilized enzymes: To develop a feasible enzymatic process for D-tagatose production, a L-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65℃ in the immobilized enzyme system, and pH 7.5 and 60℃ in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. At pH 8.0 and 60℃, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/L-h and a conversion yield of 46%. D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of the reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h^-1, 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L-h and an average conversion yield of 48 %. Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.
A double point mutant (Cys450→Ser, Asn475→Lys) from G. thermodenitrificans was selected as an enzyme producing D-tagatose due to its highest activity among the obtained enzymes. The pH and temperature for the maximal galactose isomerization reaction were pH 8.5 and 70℃ in the immobilized enzyme system. Using the immobilized enzyme, the packed-bed bioreactor produced about 160 g/L tagatose from 300 g/L galactose with an average productivity of 32 g tagatose /L-h and an average conversion yield of 48 %. The immobilized enzyme was stable for 30 days.
7. Purification of D-tagatose: After a reaction mixture of D-tagatose was treated by activated carbon to remove color, D-tagatose was separated by using MPLC with an ion exchange chromatography (Dowex 50ⅹ8, Amberlite IRA-410). The collected D-tagatose was treated by activated carbon, filtrated, concentrated, and freeze dried. From the reaction mixture of 80 g D-tagatose, 70 g of purified D-tagatose was obtained with a purification yield of 87.5%.