FOOD SCIENCE ›› 2010, Vol. 31 ›› Issue (19): 309-312.doi: 10.7506/spkx1002-6630-201019067

• Bioengineering • Previous Articles     Next Articles

Cloning and Expression of a New Thermostable Pullulanase Gene from a Deep-sea Archaeaon Strain Thermococcus siculi HJ21

WANG Shu-jun1,LU Ming-sheng1,LI Hua-zhong2,XU Jin-li1,2,JIAO Yu-liang1,FANG Yao-wei1,LIU Shu1   

  1. 1. College of Food Science and Technology, Huaihai Institute of Technology, Lianyungang 222005, China;
    2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
  • Received:2010-06-09 Revised:2010-09-14 Online:2010-10-15 Published:2010-12-29
  • Contact: WANG Shu-jun1


The gene of pullulanase was amplified from Thermococcus siculi HJ21 with degenerate primes designed based on the NCBI published conserved sequence information. The DNA sequencing and BLAST (NCBI) analysis showed that this DNA sequence was a new pullulanase gene with an open reading frame (ORF) of 4056 bp in length encoding 1351 amino acids.The gene was cloned into the expression vector, pET28a, producing a hybrid plasmid pET28a-pull. Subsequently, pET28a-pull was introduced into Escherichia coli BL21(DE3). The lysate of the transformant cells showed thermostable pullulanase activity. The SDS-PAGE analysis showed a band with apparent molecular weight of 150 kD.

Key words: Thermococcus siculi HJ21, pullulanase, gene, PCR

CLC Number: