Cloning and expression of a GH11 xylanase from Bacillus pumilus SSP-34 in Pichia pastoris GS115: Purification and characterization

Sagar Krishna Bhat Kavya Purushothaman Appu Rao Gopala Rao Appu Rao K Ramachandra Kini   

Open Access   

Published:  Jan 05, 2022


GH11 xylanase from Bacillus pumilus is a monofunctional low molecular weight single polypeptide chain xylanase with biotechnological applications. The codon-optimized xynA gene was modified to replace the signaling factor with Pichia expression system. 876 bp gene was synthesized and expressed in methylotrophic yeast Pichia pastoris GS115. The recombinant protein was purified to apparent homogeneity by conventional protein purifications methods. The specific activity, kinetic parameters Km and Vmax, temperature optima, and pH optima of the recombinant protein were similar to the native protein. The kinetic properties of native and recombinant protein indicated structural similarities of the two. The temperature stability of the recombinant protein was higher than the native protein. The half-life (t ½) at 55°C was 10.5 and 21 minutes for native and recombinant xylanase, respectively. The native GH11 xylanase had a molecular weight of 22 kDa and the recombinant protein had a higher molecular weight of 25 kDa, which could be due to glycosylation. This cloning and expression of the GH11 xylanase in P. pastoris opens the possibilities of 1) production of GH11 xylanases for industrial applications in an economical way 2) creating mutants for improved activity 3) creating mutants for improved thermal stability, and 4) desirable pH optima to meet the industrial requirements.

Keyword:     GH11 xylanase Pichia pastoris GS115 Bacillus pumilus-SSP-34 codon optimization recombinant protein purification and characterization


Bhat SK, Purushothaman K, Gopala Rao Appu Rao AR, Kini RK. Cloning and expression of a GH11 xylanase from Bacillus pumilus SSP-34 in Pichia pastoris GS115: Purification and characterization. J Appl Biol Biotech, Online First.

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text


1.Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 2005;29(1):3-23.

2. Biely P, Puls J, Schneider H. Acetyl xylan esterases in fungal cellulolytic systems. FEBS Lett 1985;186(1):80-4.

3. Cheng YF, Yang CH, Liu, WH. Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme Microb Technol 2005;37(5):541-6.

4. Paës G, Berrin JG, Beaugrand J. GH11 xylanases: structure/ function/properties relationships and applications. Biotechnol Ad 2012;30(3):564-92.

5. Wang K, Luo H, Tia J, Turunen O, Huang H, Shi, et al. Thermostability improvement of a Streptomyces xylanase by introducing proline and glutamic acid residues. Appl Environ Microbiol 2014;80(7):2158-65.

6. Singh AB. Production, characteristics, and biotechnological applications of microbial xylanases. Appl Microbiol Biotechnol 2019;103(21-22):8763-84.

7. Damas CT, Almeida MS, Kurtenbach E, Martins OB, Pereira N, Andrade CMMC, et al. Optimized expression of a thermostable xylanase from Thermomyces lanuginosus in Pichia pastoris. Appl Environ Microbiol 2003;69(10):6064-72.

8. Cregg JM, Cereghino JL, Shi J, Higgins DR. Recombinant protein expression in Pichia pastoris. Mol Biotechnol 2000;16(1):23-52.

9. He J, Yu B, Zhang K, Ding X, ChenD. Expression of endo-1, 4-beta-xylanase from Trichoderma reesei in Pichia pastoris and functional characterization of the produced enzyme. BMC Biotechnol 2009;9(56):1-10.

10. Jin X, Meng N, Xia LM. Expression of an endo-β-1,4-glucanase gene from Orpinomyces PC-2 in Pichia pastoris. Int J Mol Sci 2011;12(5):3366-80.

11. Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnol 1991;9(5):455-60.

12. Poorna CA. Purification and biochemical characterization of xylanases from Bacillus pumilus and their potential for hydrolysis of polysaccharides. Ferment Techol 2011;1(1):1-5.

13. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193(1):265-75.

14. Sebesta J, Peebles CA. Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production. Metab Eng Commun 2020;10(e0017):1-9.

15. Lammeli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970;277(5259):680-5.

16. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31(13):3406-15.

17. Subramaniyan S, Prema P. Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiol Lett 2000;183(1):1-7.

18. Zhang GM, Hu Y, Zhuang YH, Ma LX, Zhang XE. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris. Biocatal Biotransform 2006;24(5):371-9.

19. Zheng W. Enhancement of heterogeneous alkaline xylanase production in Pichia pastoris GS115. AIP Conf Proc 2017;1864(1):1- 4.

20. Teng D, Fan Y, Yang YL, Tian ZG, Luo J, Wang JH. Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Appl Microbiol Biotechnol 2007;74(5):1074-83.

21. Bretthauer RK, Castellino FJ. Glycosylation of Pichia pastorisderived proteins. Biotechnol Appl Biochem 1999;30(3):193-200.

22. Gupta, R, Brunak S. Prediction of glycosylation across the human proteome and the correlation to protein function. Pacific Symp Biocomput 2002:310-322.

23. Nakamura M, Nagamine T, Takenaka A, Aminov RI, Ogata K. Molecular cloning, nucleotide sequence and characteristics of a xylanase gene (xyn A) from Ruminococcus albus 7. Anim Sci J 2002;73(5):347-52.

24. Bhat SK, Purushothaman K, Kini KR, Rao AG. Design of mutants of GH11 xylanase from Bacillus pumilus for enhanced stability by amino acid substitutions in the N-terminal region: an in silico analysis. J Biomol Struct Dyn 2021:1-14.

Article Metrics

1 Absract views 0 PDF Downloads 1 Total views

Related Search

By author names

    Warning: Cannot modify header information - headers already sent by (output started at /home/jabonlin/public_html/jab_php/abstract.php:239) in /home/jabonlin/public_html/jab_php/articlemodule/searchArticles.php on line 1162

    Warning: Invalid argument supplied for foreach() in /home/jabonlin/public_html/jab_php/abstract.php on line 819

Citiaion Alert By Google Scholar

Name Required
Email Required Invalid Email Address

Comment required

Notice: Undefined variable: dbq35 in /home/jabonlin/public_html/jab_php/abstract.php on line 942

Warning: mysqli_num_rows() expects parameter 1 to be mysqli_result, null given in /home/jabonlin/public_html/jab_php/articlemodule/database.php on line 379