Berensmeier S, Singh S A, Meens J, Buchholz K
Department for Carbohydrates, Technical University Braunschweig, Langer Kamp 5, 38106 Braunschweig, Germany.
Appl Microbiol Biotechnol. 2004 May;64(4):560-7. doi: 10.1007/s00253-003-1446-9. Epub 2003 Dec 13.
The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.
克隆并测序了嗜碱地衣芽孢杆菌14A菌株的果胶酸裂解酶基因pelA。核苷酸序列对应于一个1026 bp的开放阅读框，该开放阅读框编码一个39个氨基酸的信号肽和一个分子量为33451 Da的成熟蛋白。成熟的PelA与属于多糖裂解酶家族1的其他果胶酸裂解酶具有显著同源性，如来自不同芽孢杆菌属和菊欧文氏菌的酶。pelA基因在大肠杆菌中表达为含有C端His标签的重组融合蛋白，可通过一步法纯化至接近均一。融合蛋白的动力学参数K(m)和Vmax值分别为0.56 g/l和51 μmol/min。在过量底物存在下，纯化的PelAHis的活性受到抑制。产物形成的表征显示不饱和三半乳糖醛酸是主要产物。进一步研究了聚半乳糖醛酸、柑橘果胶和甜菜果胶作为底物时不饱和三半乳糖醛酸的产量。
