Chen Xiaoling, Lu Wenqing, Cao Yunhe, Li Defa
National Key Laboratory of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100094, People's Republic of China.
Appl Biochem Biotechnol. 2008 May;149(2):139-44. doi: 10.1007/s12010-007-8037-7. Epub 2007 Sep 12.
Wild type (WT) DNA sequence, which encoded a mature beta-mannanase of Aspergillus sulphureus, composed of 1,152 nucleotides (nt), was amplified from pUCm-T-mann by polymerase chain reaction (PCR). Based on this DNA fragment, mutants designated as E(206)G and E(314)G were constructed by overextension PCR (OE-PCR). Glutamic acids of the 206th and 314th sites in the amino acid sequence of beta-mannanase were separately replaced by glycine in these two mutants. The WT and mutant genes were ligated into prokaryotic vector pET-28a (+) and transformed into the Escherichia coli BL21 strain, respectively. The recombinant enzyme proteins were expressed by IPTG induction and detected by Western blot. The recombinant proteins purified with Ni-NTA column were dialyzed to correctly refold. The WT recombinant beta-mannanase showed optimal activity at 50 degrees C and pH 2.4. The kinetic parameters of K (m) and V (max) for purified beta-mannanase were 1.38 mg/ml and 72.99 U/mg, respectively. However, the mutant proteins did not show any activity. It was demonstrated that E(206) and E(314) were the catalytic residues of beta-mannanase.
从pUCm-T-mann中通过聚合酶链反应（PCR）扩增出编码硫黄曲霉成熟β-甘露聚糖酶的野生型（WT）DNA序列，其由1152个核苷酸（nt）组成。基于该DNA片段，通过重叠延伸PCR（OE-PCR）构建了命名为E(206)G和E(314)G的突变体。在这两个突变体中，β-甘露聚糖酶氨基酸序列第206位和第314位的谷氨酸分别被甘氨酸取代。将野生型和突变体基因分别连接到原核载体pET-28a(+)中，并转化到大肠杆菌BL21菌株中。通过IPTG诱导表达重组酶蛋白，并通过蛋白质免疫印迹法进行检测。用Ni-NTA柱纯化的重组蛋白经透析正确复性。野生型重组β-甘露聚糖酶在50℃和pH 2.4时表现出最佳活性。纯化的β-甘露聚糖酶的米氏常数（K(m)）和最大反应速度（V(max)）动力学参数分别为1.38 mg/ml和72.99 U/mg。然而，突变体蛋白未表现出任何活性。结果表明，E(206)和E(314)是β-甘露聚糖酶的催化残基。
