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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Cloning and Characterization of a Gene Coding for a Dextransucrase from Leuconostoc mesenteroides B-742CB

Leuconostoc mesenteroides B-742CB로부터 Dextransucrase를 Coding하는 유전자 분리 및 특성 연구

  • Published : 2001.04.01
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Abstract

A gene encoding the dextransucrase(dsCB) that synthesizes mostly $\alpha-(1\rightarrow6)$ linked dextran with low amount(10%) of $\alpha-(1\rightarrow3)$ branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1 kbp DNA fragment carrying dsCB showed one open reading frame(ORF) composed of 4,536bp. The deduced amino acid sequence shows that it begins from the start codon(ATG) at position 698 of the cloned DNA fragment and extends to the termination condon(TAA) at position 5,223. The enzyme is consisted of 1,508 amino acids and has an calculated molecular mass of 168.6kDa. This calculated Mw was in good agreement with an activity band of 170kDa on non-denaturing SDS-PAGE. A recombinant E. coli DH5 $alpha$ harboring pDSCB produced extracellular dextransucrase in 2% sucrose medium, and synthesized both soluble and insoluble dextran. To compare the properties of enzyme with B-742CB dextransucrase, the acceptor reaction, hydrolysis of dextran and methylation were performed. The expressed enzyme showed the same properties as B-742CB dextransucrease, but its ability to synthesize $\alpha-(1\rightarrow3)$ branching was lower than that of B-742CB dextransucrase. In order to identify the critical amino acid residues known as conserved regions related to catalytic activity, Asp-492 was replaced with Asn. D492N resulted in a 1.6 fold decrease in specific activity.

10%의 $\alpha-(1\rightarrow3)$가지 결합을 갖고 $\alpha-(1\rightarrow6)$으로 연결된 댁스트란을 합성하는 텍스트란수크라제를 coding하는 유전자 (dsCB)를 Leuconostoc mesenteroides B-742CB로부터 분리하여 염기서열과 아미노산 서열을 결정하였다. dsCB가 포항된 6 6.lkb띄 DNA fragment는 4,536 bp의 염기로 구성된 하나의 open reading 잔ame(ORF)를 가지고 있었다. 추정된 아미노산 서열은 ORF의 698벤째 nucleotide 위치에 있는 start codon (ATG)으로부터 5,223번째 위치에 있는 slop codon(TAA)까지 였다. 구조 유전자의 아마노산은 1,5087H로 구성되고 분자량의 계산값은 168.6 kDa었고 non-denatured SDS-PAGE를 이용하여 활성 band툴 분석한 결과 170 kDa이었다. pDSCB를 까지고 있는 재조합 E. coli는 2 % sucrose배치에서 세포외로 댁스트란수크라제를 생산하였으며, soluble과 insoluble 텍스트 란을 생산하였다. 텍스트란수크라체의 효소 촉매작용에 관여 하는 것으로 얄려져있는 conserved region의 아미노산 중 Asp-492를 Asparagine으로 바꾸고자 point mutation을 시도하였고, 결과로 얻어친 D492N은 돌연변이 이전의 균에 비하여 활성이 1.6배 감소함을 확인하였다.

Keywords

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