耐热碱性磷酸酯酶基因的DNA序列分析

从栖热菌中克隆到产耐热碱性磷酸酯酶（FD－TAP）基因并进行了DNA序列分析，结果表明此2．0kb的片段含有一个1056bp的开放阅读框，编码501个氨基酸的蛋白质，其N端有一26个氨基酸的信号肽．在起始密码子的上游5bp处有一个5＇－GGAGGT－3＇的SD序列．基因编码区的（G＋C）％为68．7％，第3位密码子（G＋C）％为92．7％．FD－TAP的氨基酸序列与大肠杆菌等生物的碱性磷酸酯酶氨基酸序列比较，相同性为27％，相似性为38％．中央β－折叠区及与活性中心相关的氨基酸残基高度保守．表明FD－TAP具有与大肠杆菌碱性磷酸酯酶相似的结构和作用机制．在相当于大肠杆菌碱性磷酸酯酶的His370至His412两个金属离子结合部位之间，FD－TAP有一72个氨基酸的插入片段，提示该插入片段与FD－TAP的高耐热性相关．

Sequence Analysis of the Gene Encoding Thermostable Alkaline Phosphatase From Thermus sp. FD3041

The gene encoding thermostable alkaline phosphatase (FD-TAP) of an extreme thermophile, Thermus sp. FD3041, was cloned and sequenced. This gene consisted of a single open reading frame(ORF) of 1 506 bp, preceded by a putative Shine-Dalgarno ribosome binding site (5'-GGAGGT-3'). The G+C content of the coding region was 67. 8% and that of the third nucleotide of the codons was 92. 7%. The ORF corresponded to a protein of 501 amino acid residues with a calcultated molecular mass of 54 676. A comprison of the amino acid sequence of FD-TAP with that E. colt AP showed that the residue identity was 27%,similarity was 38%. The residues in catalytic site and mental ligand were highly conserved,indicating that FD-TAP had a similar tertiary structure and possibly a similer catalytic mechanism to that of E. colt AP. The presence of 72 extra residues between two metal ligand (corresponding to His 327 and His412 of E. colt AP) may play an important role to the high thermostability of FD-TAP.