College of Marine Life Sciences, Ocean university of China, Yushan Road, No. 5, Qingdao, China.
College of Marine Life Sciences, Ocean university of China, Yushan Road, No. 5, Qingdao, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266003 Qingdao, China.
Int J Biol Macromol. 2018 Oct 1;117:727-734. doi: 10.1016/j.ijbiomac.2018.05.235. Epub 2018 Jun 1.
A high molecular weight (Mw) pullulan has many potential applications in various fields. α-Amylase, glucoamylase and pullulanase were thought to play an important role in high Mw pullulan biosynthesis. However, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and characterized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could produce 46.2 g/L of pullulan with a Mw of 3.02 × 10 Da and grown in the 10-L fermentor could yield 58.14 g/L of pullulan with the same Mw while its wild type strain P16 produced 65.5 ± 3.5 g/L of pullulan with a Mw of 0.35 × 10 Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan.
高分子量（Mw）普鲁兰在各个领域都有许多潜在的应用。α-淀粉酶、糖化酶和普鲁兰酶被认为在高 Mw 普鲁兰生物合成中发挥重要作用。然而，目前还没有遗传证据支持这一作用。在本研究中，从产高普鲁兰酵母 Aureobasidium melanogenum P16 中克隆了编码α-淀粉酶、糖化酶和普鲁兰酶的基因，并对其进行了鉴定。从克隆的α-淀粉酶基因、糖化酶基因和异普鲁兰酶基因推导的蛋白分别具有相应的保守氨基酸序列，而不是普鲁兰酶基因。当它们的单个基因缺失后，单个突变体产生的普鲁兰的 Mw 大大增加，普鲁兰的浓度降低。研究发现，在摇瓶水平生长的三突变体 DT15 可以产生 46.2 g/L 的普鲁兰，Mw 为 3.02×10 Da，在 10 L 发酵罐中生长可以产生 58.14 g/L 的普鲁兰，Mw 相同，而其野生型菌株 P16 可以产生 65.5±3.5 g/L 的普鲁兰，Mw 为 0.35×10 Da。当这些基因被互补后，普鲁兰的产量、所产生普鲁兰的 Mw 等都得到了恢复。所有结果表明，α-淀粉酶、糖化酶和异普鲁兰酶确实可以决定所产生普鲁兰的 Mw。
