Abstract：Starch is one kind of abundant biomass resources which is widely used in lots of industries such as food, brewing and pharmaceutical industries. Pullulanase (pulA, EC 3.2.1.41) is one of the starch-debranching enzymes which catalyzes the hydrolysis of α-1,6 glycosidic bonds in starch, and improves the utilization of starch. Hence, it has a more profound reason to develop pullulanase in our own way. In the previous study, a pullulanase high-producing strain, Klebiella variicola strain 7, was obtained from the soil near a starch factory. The gene encoding pullulanase (GenBank No. KJ146839) from Klebiella variicola strain 7 was expression in Escherich coli BL21(DE3). In order to improve expression quantity, secretion efficiency and properties of pullulanase, Mutants of M1 and M2 were built using the method of genetic engineering of removing N- terminal amino acids of pullulanase to improve expression quantity, secretion efficiency and properties of pullulanase. The results showed that, the optimum temperature of both M1 and M2 were 45 ℃, and the optimum pH of M1 was 6.0, while the optimum pH of M2 was 5.6. The half-life and specific activity of M2 were 37 minutes and 582.204 U/mg, which were 6.17- and 1.6- fold that of M1. With pullulan as substrate, Kinetic studies showed that the Vmax, Kcat, Km and Kcat /Km of M2 was 0.001 3 μmoL/(mL·s)-1, 191.80 s-1, 0.30 mg/mL, 693.30, respectively. Compared with M1, the substance affinity increased, and the catalytic efficiency was 2- fold that of M1. This study provides a new way and thinking for improving the specific activity and half-life period of enzymes by N-terminal amino acids truncation.
[1]Nair S U, Singhal R S, Kamat M Y.Induction of pullulanase production in Bacillus cereus FDTA-13[J]. Bioresource Technol, 1998, 4: 856-859.[J].Bioresource Technoledge, 1998, 4(2007):856-859
[2]Jiao Y L, Wang S J, Lv M S, et al.A GH57 family amylopullulanase from deep-sea Thermococcus siculi: expression of the gene and characterization of the recombinant enzyme [J]．Curr．Microbiol, 2011, 62：222-228．[J].Current Microbiology, 2011, 62(2011):222-228
[3]Mathupala S P, Lowe S E, Podkovyrov S M, et al.Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E,and identification of the active site by site-directed mutagenesis[J].J Biol Chemi, 1993, 268(22):16332-16344
[4]Bertoldo C, Antranikian G.Starch-hydrolyzing enzymes from thermophilic archaea and bacteria[J].CurrOpin ChemBio, 2002, 6(2):151-160
[5]Singh R S, Saini G K, Kennedy J F.Covalent immobilization and thermodynamic characterization of pullulanase for the hydrolysis of pullulan in batch system[J].Carbohydrate Polymers, 2010, 81(2):252-259
[6]Kuroiwa T, Shoda H, Ichikawa S, et al.Immobilization and stabilization of pullulanase from Klebsiella pneumoniae by a multipoint attachment method using activated agar gel supports[J].Process Biochemistry, 2005, 40(8):2637-2642
[7]朱俊晨, 王小菁.酶的分子设计、改造与工程应用[J].中国生物工程志, 2004, 24(8):32-37
[8]Teague W M, Brumm P J, Allen L N, et al.Pullulanase expression constructs containing α-amylase promoter and leader sequences[P]. USA, 2001, 6300115B1.
[9]Duan X G, Wu J.Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation. China, 2015, 81: 1926-1931.[J].Applied and Environmental Microbiology, 2015, 81(6):1926-1931
[10]张雨杭, 聂慧慧, 焦国宝, 等.克雷伯氏菌菌株- 普鲁兰酶基因的克隆及酶学性质研究[J].农业生物技术学报, 2015, 23(12):1632-1638
[11]陈文波.普鲁兰酶的产生菌筛选及其表达与分泌调控[D]. 博士学位论文, 江南大学, 2013, 51-61.
[12]Sauvonnet N, Pugsley A P.Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting β-lactamase secretion by the general secretory pathway[J].Mol Microbiol, 1996, 22(1):1-7
[13]叶延欣, 闫鹏飞, 胡渝, 刘亮伟.木聚糖酶二级结构含量对热稳定性的影响[J].河南农业大学学报, 2013, 47(4):446-450
