文献类型：期刊文献

英文题名：A novel cold-adapted type I pullulanase of Paenibacillus polymyxa Nws-pp2: in vivo functional expression and biochemical characterization of glucans hydrolyzates analysis

作者：Wei, Wei[1];Ma, Jing[1];Chen, Si-Qi[1];Cai, Xiang-Hai[1];Wei, Dong-Zhi[1]

机构：[1]E China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2015

卷号：15

期号：1

外文期刊名：BMC BIOTECHNOLOGY

收录：;EI(收录号：20154301428728);WOS:【SCI-EXPANDED(收录号:WOS:000363016900001)】；

基金：This research was financially supported by the National High Technology Research and Development Program of China (No. 2013AA102109, NO. 2012AA022206), the National Natural Science Foundation of China (No. C050203-31200596, 31570795), the National Major Science and Technology Projects of China (No. 2012ZX09304009) and the National Basic Research Program of China (973, Program No. 2012CB721103).

语种：英文

外文关键词：Pullulanase type I; Paenibacillus polymyxa; Cold-adapted pullulanase; Functional expression; Enzymatic properties; Starch hydrolysis products

摘要：Background: Pullulanase is an important debranching enzyme and has been widely utilized to hydrolyse the alpha-1,6 glucosidic linkages in starch/sugar industry. Selecting new bacterial strains or improving bacterial strains is a prerequisite and effective solution in industrial applications. Although many pullulanase genes have been cloned and sequenced, there is no report of P. polymyxa type I pullulanase gene or the recombinant strain. Meanwhile most of the type I pullulanase investigated exhibit thermophilic or mesophilic properties. There are just few reports of cold-adapted pullulanases, which have optimum activity at moderate temperature and exhibit rather high catalytic activity at cold. Previously, six strains showing distinct pullulan degradation ability were isolated using enrichment procedures. As containing novel bacterium resource and significant pullulanase activity, strain Nws-pp2 was selected for in-depth study. Methods: In this study, a type I pullulanase gene (pulN) was obtained from the strain P. polymyxa Nws-pp2 by degenerate primers. Through optimization of induced conditions, the recombinant PulN achieved functional soluble expression by low temperature induction. The enzyme characterizations including the enzyme activity/stability, optimum temperature, optimum pH and substrate specificity were also described through protein purification. Results: The pullulanase gene (named pulN), encoding a novel cold-adapted type I pullulanase (named PulN), was obtained from isolated strain Paenibacillus polymyxa Nws-pp2. The gene had an open reading frame of 2532-bp and was functionally expressed in Escherichia coli through optimization of induced conditions. The level of functional PulN-like protein reached the maximum after induction for 16 h at 20 degrees C and reached about 0.34 mg/ml (about 20 % of total protein) with an activity of 6.49 U/ml. The purified recombinant enzyme with an apparent molecular mass of about 96 kDa was able to attack specifically the alpha-1,6 linkages in pullulan to generate maltotriose as the major product. The purified PulN showed optimal activity at pH 6.0 and 35 degrees C, and retained more than 40 % of the maximum activity at 10 degrees C (showing cold-adapted). The pullulanase activity was significantly enhanced by Co2+ and Mn2+, meanwhile Cu2+ and SDS inhibited pullulanase activity completely. The Km and Vmax values of purified Pul(N) were 15.25 mg/ml and 20.1 U/mg, respectively. The PulN hydrolyzed pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity and products analysis proved that the purified pullulanase from Paenibacillus polymyxa Nws-pp2 belong to a type I pullulanase. Conclusions: This report of the novel type I pullulanase in Paenibacillus polymyxa would contribute to pullulanase research from Paenibacillus spp. significantly. Also, the cold-adapted pullulanase produced in recombinant strain shows the potential application.