深海细菌太平洋火色杆菌中α-淀粉酶基因amy608的克隆表达及其酶学性质研究  

Cloning,expression and characterization of α-amylase gene amy608 from deep-sea bacterium Flammeovirga pacifica

作　　者：商谢谢 蔡亚萍[3] 金敏[1,2] 曾润颖[1,2] 

机构地区：[1]国家海洋局第三海洋研究所海洋生物遗传资源省部共建国家重点实验室培育基地,福建厦门361005 [2]福建省海洋生物资源开发利用协同创新中心,福建厦门361005 [3]厦门出入境检验检疫局检验检疫技术中心,福建厦门361100

出　　处：《应用海洋学学报》2016年第1期130-137,共8页Journal of Applied Oceanography

基　　金：中国大洋矿产资源研究开发协会资助项目(DY125-15-T-06);厦门南方海洋中心资助项目(13GZP003NF09);国家微生物资源平台资助项目(NIMR-2014-9)

摘　　要：从来自深海沉积物的太平洋火色杆菌（Flammeovirga pacifica）的基因组中发现了一个全长为1 875 bp的α-淀粉酶基因amy608.该基因在数据库中找不到具有同源性的序列,而所编码的Amy608蛋白与已注册蛋白的氨基酸序列相似性最高仅为56%,但具有α-淀粉酶水解活性所必需的保守基序DXEXD.进化树分析表明其属于糖苷水解酶13（GH13）家族第二亚家族.构建了p ColdΙ-amy608表达载体,在大肠杆菌中进行重组Amy608蛋白的异源表达,并采用镍离子亲和层析柱对重组蛋白进行了纯化.酶学性质分析表明,重组酶Amy608的最适作用温度为40℃,在40℃保温4 d后,仍保留70%以上的酶活,显示出良好的中温热稳定性;最适p H值为7.0,p H值范围在6~9时仍保留60%以上的酶活力,表明该酶具有较宽的p H值作用范围.Ca^2＋、Na^＋、K^＋对α-淀粉酶Amy608有激活作用,尤其是Ca2＋可使酶活显著提高40%.薄层色谱分析结果显示该酶水解可溶性淀粉的最终产物以葡萄糖为主,表明该酶是一个糖化型淀粉水解内切酶.这些结果表明Amy608为GH13家族第二亚家族中一个新型的α-淀粉酶.A 1 875 bp α-amylase gene（ designated as amy608） was identified from Flammeovirga pacific,which was isolated from deep-sea sediments. The DNA sequence of amy608 exhibited no significant similarity to those of any known genes. Amino acid sequence analysis showed that Amy608 possess a conserved DXEXD motif,which is essential for the hydrolysis activities of α-amylase. However,Amy608 shares only 56% maximum amino acid sequence identity to other existing α-amylases. According to the phylogeny analytical results,Amy608 could be further classified into GH13（ glycoside hydrolase 13） subfamily-2. After heterologous expression in Escherichia coli,the recombinant Amy608 was purified with a Ni^ ＋ affinity column. The recombinant enzyme was most active at 40℃and retained more than 70% of its initial activity after incubation at 40℃ for 4 days,exhibiting a good thermostability at medium temperatures. The optimal p H for Amy608 was determined to be 7 and Amy608 could adapt to wide p H environments as it retained more than 60% of its initial activity in p H 6 ~ 9 environments. Some ions（ Ca^2 ＋、Na^＋、K^＋）,especially Ca^2 ＋could largely increase the activity of Amy608 by up to 40%. Thin layer chromatography results showed that Amy608 can hydrolyze starch into glucose in a time dependent manner,suggesting that Amy608 is a saccharogenic type endo-enzyme with starch hydrolysis activities. Collectively,our study presents a novel GH13 subfamily-2 α-amylase.

关 键 词：海洋生物学 太平洋火色杆菌 Α-淀粉酶 中温稳定性 深海 

分 类 号：P735[天文地球—海洋生物学]