Journal of agriculture & life science (농업생명과학연구)

Institute of Agriculture & Life Science, Gyeongsang National University (경상대학교 농업생명과학연구원)
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Cloning and Expression of a Alkaline Protease from Bacillus clausii I-52

Bacillus clausii I-52로부터 alkaline protease 유전자의 클로닝 및 발현

  • 주한승 (씨앤제이바이오텍(주)) ;
  • 최장원 (대구대학교 바이오산업학과)
  • Received : 2011.11.07
  • Accepted : 2011.12.20
  • Published : 2011.12.30
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Abstract

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

인천 연안의 심하게 오염된 갯벌로부터 강력한 세포외 알카리성 단백질 분해효소를 생산하는 호알카리성 Bacillus clausii I-52를 분리하였으며, 이 균주로부터 알카리성 단백질 분해효소의 유전자를 cloning하여 서열 분석을 하였다. Chromosome 서열이 완전히 밝혀진 Bacillus subtilis의 서열을 기초로 하여 알카리성 단백질 분해효소 및 promoter를 포함하도록 primer를 고안하여 PCR을 수행하여 2,277 bp의 DNA 단편을 얻었으며 BLAST 분석 결과 29 개의 아미노산으로 이루어진 signal peptide, 77 개의 아미노산으로 이루어진 propeptide 및 275 개의 아미노산을 갖는 활성형의 BCAP으로 구성된 총 381 개의 아미노산을 코딩하는 1,143 bp의 open reading frame을 확인하였다. 활성형 BCAP의 N-말단 아미노산은 Ala이며, 분자량 및 pI 값은 각각 27698.7 Da과 6.30으로 계산되었다. 아미노산 상동성을 분석한 결과, B. subtilis 유래의 nattokinase precursor 및 B. subtilis BSn5 유래의 subtilisin E precursor와 99%의 서열 상동성을 나타내어 B. clausii I-52 유래의 BCAP은 subtilisin 계열의 단백질 분해효소임을 확인하였다. E. coli BL21(DE3)에서 발현한 재조합 BCAP는 N-Suc-Ala-Ala-Pro-Phe-pNA 를 효율적으로 분해하였다. Refolding한 재조합 BCAP은 전형적인 serine protease inhibitor인 PMSF에 의하여 강하게 효소 활성이 억제됨으로써 serine protease 계열의 단백질 분해효소임을 알 수 있었다.

Keywords

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