Erden-Karaoğlan Fidan, Karakaş-Budak Barçın, Karaoğlan Mert, Inan Mehmet
Department of Food Engineering, Erzincan Binali Yildirim University, Erzincan, Turkey.
Department of Food Engineering, Akdeniz University, Antalya, Turkey.
Protein Expr Purif. 2019 Oct;162:83-88. doi: 10.1016/j.pep.2019.05.008. Epub 2019 May 25.
In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter utilizing the Saccharomyces cerevisiae α-mating factor sequence for extracellular secretion. The molecular weight of the recombinant enzymes BK07pul and PY22pul were both approximately 90 kDa. Both enzymes showed highest activity at 40 °C, however PY22pul showed optimum activity at pH 6 whereas, BK07pul had highest activity at pH 8. BK07pul and PY22pul activities were determined as 8.46 U/mL and 15 U/mL. The enzyme stability of BK07pul was higher (89%) than PY22pul (68%) where relative activity was determined as activity remaining after 1 h at corresponding optimum conditions for each. Amino acid homology evaluation revealed the two enzymes had 80% identity in primary structure. The presence of conserved sequences consisting of 7 amino acids (YNWGYDP) in both enzymes confirmed these to be type I pullulanases, capable of hydrolyzing α-1,6 glucosidic bonds of pullulan resulting in maltotriose units.
在本研究中，将野生分离株枯草芽孢杆菌BK07和枯草芽孢杆菌PY22（源自枯草芽孢杆菌168的突变株）的支链淀粉酶基因转化到巴斯德毕赤酵母KM71H中。利用酿酒酵母α-交配因子序列进行细胞外分泌，在AOX1启动子的调控下通过甲醇诱导实现细胞外重组蛋白的产生。重组酶BK07pul和PY22pul的分子量均约为90 kDa。两种酶在40°C时均表现出最高活性，然而PY22pul在pH 6时表现出最佳活性，而BK07pul在pH 8时具有最高活性。BK07pul和PY22pul的活性分别测定为8.46 U/mL和15 U/mL。BK07pul的酶稳定性（89%）高于PY22pul（68%），相对活性定义为在各自相应最佳条件下1小时后剩余的活性。氨基酸同源性评估显示这两种酶的一级结构具有80%的同一性。两种酶中均存在由7个氨基酸（YNWGYDP）组成的保守序列，证实它们为I型支链淀粉酶，能够水解支链淀粉的α-1,6糖苷键，产生麦芽三糖单元。
