Thermostable Phospholipase A1 Mutants and Process forPreparing the Same

본 발명은 인지질 분해/합성 효소인 포스포리파제 A1의 돌연변이체로서 내열성을 가지는 포스포리파제 TA3 및 TA13, 그를 암호화하는 유전자, 전기 돌연변이체의 유전자를 포함하는 재조합 발현벡터로 형질전환된 미생물 및 그로부터 포스포리파제 A1의 돌연변이체를 제조하는 방법에 관한 것이다. 본 발명의 돌연변이체는 돌연변이 유발형 중합효소연쇄반응(mutagenic PCR)에 의하여 유발된 포스포리파제 A1의 돌연변이체의 유전자를 포함하는 벡터로 형질전환된 대장균주를 배양한 후, 이로부터 포스포리파제 A1의 돌연변이체를 분리 및 정제하는 단계를 포함하는 방법에 의하여 제조할 수 있다. 본 발명의 포스포리파제 A1의 돌연변이체는 야생형의 포스포리파제 A1에 비하여 열에 대한 안정성이 향상되었을 뿐만 아니라, 고온에서의 효소활성 자체도 증가하였으므로, 생물공정, 의약, 화장품 및 식품 등에 유용하게 활용될 수 있을 것이다. 세라시아 속(Serratia sp.), 포스포리파제 A1, 멤브레인 결합형 활성탐

방향성 진화를 이용한 phospholipase A1의 열안정성과 유기용매 내성의 증가

학위논문(박사) - 한국과학기술원 : 생물과학과, 2000.2, [ viii, 120 p. ]The thermal stability and catalytic activity of phospholipase $A_1$ from Serratia sp. MK1 were improved by an evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR to introduce random mutations and filter-based screening of the resultant mutant library, and identified as having six (mutant TA3) and seven (mutant TA13) amino acid substitutions, respectively. Different types of the substitutions were found in two mutants, resulting in the increase of nonploar residues (mutant TA3) or changes between side chains within polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on their stability and catalytic activity was investigated. The $T_m$ values of TA3 and TA13 were increased by 7 and 11℃, respectively. Compared to the wild-type enzyme, the temperature optimum for activity was about 10℃ higher and the specific activity was significantly higher at the temperature range examined. The stability and activity of phospholipase $A_1$ were also enhanced by the same approach that used for thermostability evolution, except that the positive nine variants from the first round (30% DMSO, 6 h) were in vitro recombined by DNA shuffling in preparing the second library. Three mutants (SA8, SA17 and SA20) were isolated after exposure to 50% DMSO for 36 h and activity assay on the screening gel containing 30% DMSO. Mutants SA8, SA17 and SA20 were 3.0, 3.9, 2.6-fold more stable in 50% DMSO and 4.6, 5.5, 4.0-fold more active in 30% DMSO than the wild-type phospholipase $A_1$. Three mutants were also more stable in all organic solvents tested, compared with the wild-type enzyme. Thus, evolutionary molecular engineering was found to be an effective and efficient approach to increasing stability without compromising enzyme activity. Moreover, the more stable and more active enzymes could be obtained when the pressures on two properties (i.e. stability and ac...한국과학기술원 : 생물과학과

Serratia sp. MK1으로부터 Phospholipase A1A_1 유전자의 Cloning 및 대장균에서의 발현

학위논문(석사) - 한국과학기술원 : 생물과학과, 1996.2, [ iv, 43 p. ]Phospholipase $A_1$ gene from Serratia sp. MK1 was cloned in Escherichia coli DH5$\alpha$. The presence of two open reading frames of 966 and 705 nucleotides was found from nucleotide sequences of cloned gene. Two open reading frames were designated as phlA and phlB, respectively. First open reading frame, phlA, encoded a 33.4 kDa polypeptide of 321 amino acid residues, which was designated PLA. The start codon of second open reading frame was 5 bases upstream of stop codon of phlA. Second open reading frame, phlB, encoded a 25.7 kDa polypeptide of 234 amino acid residues, which was designated PLB. The gene structure and deduced amino acid sequences revealed high homology to those of Serratia liquefaciens. When E.coli containing pTrAB was cultured at $37^\circ C$, phospholipase $A_1$ was formed about 20\% of total proteins for 4 hours after induction at final 0.8mM IPTG. Moreover, phospholipase $A_1$ activity from supernatant was also detected on selective agar plates. It was phlA gene product (PLA) that had phospholipase $A_1$ activity. Expressed phlA gene product was detected in both cells and supernatant, as a result of secretion through a mechanism in which was involved by phlB gene product, or cell lysis by phospholipase $A_1$ activity degrading cell membrane. Though a portion of this phospholipase $A_1$ was secreted or leaked into the culture medium from E.coli host cells, the rest of overexpressed phospholipase $A_1$ were intracellular as enzymatically inactive and insoluble protein. Overexpression of cloned phospholipase $A_1$ is toxic to the host and may be resulted in problems of cell lysis and expression plasmid instability.한국과학기술원 : 생물과학과