A Novel GH10 Xylanase Xyn13-3 from Alkaline Soil: Gene Cloning and Heterogenous Expression
Xylanase, as critical hemicellulose degrading enzyme, it could degrade xylan into oligosaccharides and has been widely used in medicine, paper-making, animal forage, marine functional foods and textile industry. So exploring new xylanase with excellent en
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1 Introduction Hemicelluloses are the second most abundant plant polysaccharides in nature. Due to their potential role as sustainable energy sources, hemicelluloses are increasingly becoming an important concern [1]. The main chain of xylan is constituted with xylose unit through the b-1,4-linkage, and it is usually substituted with varying levels of L-arabinofuranosyl, galactosyl, acetyl, glucuronyl, and 4-O-methylglucuronyl groups [2]. Among them, xylanase plays a crucial role in the hydrolysis of the xylan back-bone [3], in which xylanase cleaves the b-1,4-glycosidic bond between xylose residues to release xylooligosaccharides. A great potential for some biotechnological applications in many biotechnological application especially in food, bioconversion, pulp and paper industries [4]. Due to the increasing demand for xylanase production, exploring new enzyme sources has been a hot topic in recent years [5]. Therefore, we tried to explore some novel alkaline xylanase from alkaline soil, in this study, a novel xylanase of family 10 (Xyn13-3) was obtained from the metagenomic DNA of alkaline soil and it was heterologously expressed in Pichia pastoris GS115.
Haiyan Qiu and Zhongyuan Li: Co-first authors. H. Qiu Z. Li H. Wang S. Li T. Zhang (&) College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2018 H. Liu et al. (eds.), Advances in Applied Biotechnology, Lecture Notes in Electrical Engineering 444, https://doi.org/10.1007/978-981-10-4801-2_10
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2 Materials and Methods 2.1
Strains, Plasmids, and Culture Conditions
E. coli DH5a and the vector PMD-19T (TaKaRa, Dalian, China) were used for gene cloning. Pichia pastoris GS115 and vector pPIC9 used for gene expression were purchased from Invitrogen (Carlsbad, CA, USA). Reagents and chemicals used for DNA manipulation were purchased from ThermoFisher Scientific (Shanghai, China).Yeast Nitrogen Base (YNB) with amino acids was purchased from Solarbio (Beijing, China). All yeast culture media were prepared according to the Yeast Protocols Handbook.
2.2
Alkaline Soil Sampling and Metagenomic DNA Extraction
Alkaline soil was obtained from the coastal saline area in Tianjin (China) and stored in −20 °C. The pH was measured by pH meter (SevenEasy, Shanghai). About 1 g soil sample was firstly grinded by liquid nitrogen, and the metagenomic DNA was extracted by CTAB-SDS method [6]. The crude DNA was purified by DNA purification kit (Solarbio, Beijing, China), and was detected by nucleic acid electrophoresis.
2.3
Cloning and Sequence Analysis of Xylanase Gene Xyn13-3
The xylanase gene Xyn13-3 was obtained by Touch-down PCR and TAIL-PCR methods. Firstly, the conserved sequence of Xyn13-3 was amplificated by Touch-down PCR with primers GH10F and GH10R according to Wang et al. [7]. The Nucleotide fragment was sequenced by GENEWIZ, Beijing, China. The full gene sequence was amplificated using Tail-PCR method with the six s
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