微生物學(xué)通報(bào) OCT 20,2010,37(10):1427～1431

一種不依賴鈣離子的酸性α淀粉酶基因的克隆表達(dá)

楊鍵1,2  王青艷1,2  金輝1,2  秦艷2  王成華2,3  黃日波1,2*

(1. 廣西大學(xué)生命科學(xué)與技術(shù)學(xué)院 廣西亞熱帶生物資源保護(hù)利用重點(diǎn)實(shí)驗(yàn)室  廣西 南寧  530005)

(2. 廣西科學(xué)院國(guó)家非糧生物質(zhì)能源研究工程中心  廣西 南寧  530007)

(3. 南京工業(yè)大學(xué)生物與制藥工程學(xué)院  江蘇 南京  210009)

摘  要: 以產(chǎn)酸性淀粉酶菌株Bacillus sp. CN7的基因組DNA為模板, PCR擴(kuò)增到 淀粉酶成熟肽基因, 將該基因插入表達(dá)質(zhì)粒pSE380中, 構(gòu)建重組質(zhì)粒pSE380-cn7a,。將重組質(zhì)粒導(dǎo)入到Escherichia coli JM109中, IPTG誘導(dǎo)表達(dá),。重組酶經(jīng)Sephacryl S300,、Ni-NTA純化后測(cè)定其酶學(xué)性質(zhì),。重組酶CN7A的最適溫度為65°C, 最適pH為5.5?6.0, 對(duì)可溶性淀粉的Km值為3.784 g/L, 最大反應(yīng)速度為101.2 mg/(L·min), 該酶的熱穩(wěn)定性不依賴鈣離子,。

關(guān)鍵詞: 酸性淀粉酶, 不依賴鈣離子, 克隆表達(dá), pKa

Cloning and Expression of Ca-independent Acid α-amylase Gene

YANG Jian1,2  WANG Qing-Yan1,2  JIN Hui1,2  QIN Yan2  WANG Cheng-Hua2,3

HUANG Ri-Bo1,2*

(1. Guangxi Key Laboratory of Subtropical Bio-resource Conservation and Utilization, College of Life Science and Technology, Guangxi University, Nanning, Guangxi 530005, China)

(2. National Non-grain Bio-energy Engineering Research Center, Guangxi Academy of Sciences, Nanning,Guangxi 530007, China)

(3. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing,Jiangsu 210009, China)

Abstract: The  -amylase gene cn7a was amplified by PCR from Bacillus sp. CN7 genome DNA. The recombinant plasmid pSE380-cn7a was constructed by inserting gene cn7a into expression vector pSE380 and then transformed into Escherichia coli JM109. The purified amylase CN7A showed an op-timal activity at pH 5.5?6.0 and 65°C, the Km value is 3.784 g/L taking soluble starch as substrate, and the maximum velocity was determined as 101.2 mg/(L·min). Ca ion was not required for the thermal stability of CN7A.

Keywords: Acid amylase, Ca-independent, Cloning and expression, pKa

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