UV mutagenesis for the overproduction of xylanase from Bacillus mojavensis PTCC 1723 and optimization of the production condition

Document Type : Original Article


1 Department of Microbiology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Microbiology, College of Basic Science, Islamic Azad University, Tehran North Branch, Tehran, Iran

3 Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran

4 Department of Biotechnology, College of Basic Science, Tarbiyat Modares University, Tehran, Iran


Objective(s):[p1]  This study highlights xylanase overproduction from Bacillus mojavensis via UV mutagenesis and optimization of the production process.
Materials and Methods:Bacillus mojavenis PTCC 1723 underwent UV radiation. Mutants’ primary screening was based on the enhanced Hollow Zone Diameter/ Colony Diameter Ration (H/C ratios) of the colonies in comparison with the wild strain on Xylan agar medium. Secondly, enzyme production of mutants was compared with parental strain. Optimization process using lignocellulolytic [AGA2] wastes was designed with Minitab software for the best overproducer mutant.
Results: H/C ratio of 3.1 was measured in mutant number 17 in comparison with the H/C ratio of the parental strain equal to 1.6. Selected mutant produced 330.56 IU/ml xylanase. It was 3.45 times more enzyme than the wild strain with 95.73 IU/ml xylanase. Optimization resulted 575 IU/ml xylanase, with wheat bran as the best carbon source, corn steep liquor as the best nitrogen source accompanied with natural bakery yeast powder, in a medium with pH 7, after 48 hr incubation at 37°C, and the shaking rate of 230 rpm. Optimum xylanase activity was assayed at pH 7 and 40°C. Enzyme stability pattern shows it retains 62% of its initial activity at pH 9 after 3 hr. It also maintains up to 66% and 59% of its initial activity after 1 hr of pre-incubation at 70°C and 80°C.
Conclusion: Mutation and optimization caused 5.9 times more enzyme yield by mutant strain. Also this enzyme can be categorized as an alkali-tolerant and thermo-stable xylanase.


1.Collins T, Gerday C, Feller G. Xylanase, Xylanase families and extermophilic xylanses. FEMS Microbiol Rev 2005; 29:3-23.
2.Schallmey M, Singh A, Ward OP. Developments in the use of Bacillus species for industrial production. Can J Microbiol 2004; 50:1-17.
3.Zofia OB. Xylanase from Bacillus subtilis expressed in Bacillus subtilis, Chemical and Technical assessment (CTA). 63rd JECFA? 2004; 1-5.
4.Haki GD, Rakshit SK. Developments in industrially important thermostable enzymes: a review. Bioresource Technol 2003; 89:17-34.
5.Terrasan CRF, Temer B, Sarto C, Silva Junior FG, Carmona EC. Xylanase and β-xylosidase from Penicillium janczewskii: production, physico-chemical properties, and application of the crude extract to pulp biobleaching. BioResources 2013; 8:1292-1305.
6.Butt MS, Tahir-Nadeem M, Ahmad Z. Sultan T. Xylanase and their application in baking industry. Food Technol Biotechnol 2008; 46:22-31.
7.Abdel-Aziz MS, Talkhan FN, Fadel M, Abou Zeied AA, Abdel-Razik AS. Improvement of xylanase production from Streptomyces Pseudogriseolus via UV mutagenesis. Aus J Basic Appl Sci 2011; 5:1045-1050.
8.Dwivedi P, Vivekanand V, Ganguly R. Singh RP. Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation. Biomass Bioenergy J 2009; 33:581-588.
9.Porsuk I, Özakin S, Balİ B, İnce Yilmaz E. A cellulase-free, thermoactive, and alkali xylanase production by terrestrial Streptomyces sp. CA24. Turk J Biol 2013; 37:370-375.
10.Sugumaran KR, Kumar BK, Mahalakshmi M, Ponnusami V. Cassava bagasse-low cost substrate for thermo-tolerant xylanase production using Bacillus subtilis. Int J Chem Tech Res 2013; 5:394-400.
11.Usama FA, Ibrahim ZM, Georg I. Ethanol and xylitol production from xylanase broth of Thermomyces lanuginosus grown on some lignocellulosic wastes using Candida tropicalis EMCC2. Life Sci J 2013; 10:968-978.
12.Sharma N, Ranchan SH, and Pathania SH. Production, purification and characterization of cellulose free- xylanase by Bacillus coagulans B30 using lignocellulosic forest wastes with different pretreatment methods. J Agroalimentary Processes  Technologies 2013; 19:28-36.
13.Gupta G, Sahai V, Gupta RK. Optimization of xylanase production from Melanocarpus albomyces using wheat straw extract and its scale up in stirred tank bioreactor. Indian J Chem Technol 2013; 20:282-289.
14.Coman G, and Bahrim G. Minimizing cellulase biosynthesis from cellulase–free xylanase production with Streptomyces ssp. P12-137 using optimization by response surface methodology. Cell Chem Technol 2011; 45:245-250.
15.Abo-State MAM, Ghaly MF, Abdellah EM. Optimization of cellulase(s) and xylanase production by thermophilic and alkaliphilic Bacillus isolates. Am Eur J Agric Environ Sci 2013; 13:553-564.
16.Tork sanaa, Aly MM, Alakilli SY, Al-Seeni MN. Production and characterization of thermostable xylanase from Bacillus subtilis XP10 isolated from marine water. Afr J Biotechnol 2013; 12:780-790.
17.Loera corral O, Villasenor-Ortega F. Xylanases, advances in agricultural and food biotechnology. Enzyme Biotechnol 2006; 2:305-322.
18.Haq I,  Hussain R, Hameed U, Javad M. Selection of Aspergillus niger mutant using antimetabolite 2-deoxy D-glucose after N-methyl N-nito N-nitroso guanidine(MNNG) treatment. Pak J Botany؟ 2008; 40:2613-2623.
19.Tasneem M, Khan A, Ashraf H, Haq I. Xylanase Biosynthesis by chemically mutated strain of Aspergillus niger. J Food Technol 2003; 1:178-181.
20.Rahim T, Ray AL, Beauty SP, Gomes DJ. Induction of mutation in Neurospora crassa with Ultraviolet radiation and evaluation of cellulase and xylanase activities. Bangladesh J Bot 2009; 3:201-203.
21.Korbekandi H, Darkhal P, Hojati Z, Abedi D, Hamedi J, Pourhosein M. Overproduction of clavulanic acid by UV mutagenesis of Streptomyces clavuligerus. Iran J Pharm Res 2010; 9:177-181.
22.Kapoor M, Nair LM, Kuhad RC. Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora. Biochem Eng J 2008; 38:88-97.
23.Akhavan Sepahi A, Ghazi SH, Akhavan Sepahi M. Cost effective production and optimization of alkaline xylanase by indigenous Bacillus mojavensis AG137 fermented on agricultural waste. Enzyme Res 2011; 10:1-9.
24.Sa-Pereira P, Mesquita A, Duarte JC, Aires Barros MR, Costa-Ferreira M. Rapid production of thermostable Cellulase-free xylanase by a strain of Bacillus subtilis and its properties. Enzyme Microb Tech 2002; 30:924-933.
25.Varalakshmi KN, Kumudini BS, Nandini BN, Solomon JD, Mahesh B, Suhas R. Characterization of alpha Amylase from Bacillus sp.1 isolated from paddy seeds. J Appl Biosci 2008; 1:46-53.
26.Bailey MJM, Biely P, Poutanen k. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 1992; 23:257-270.
27.Miller GL. Use of Dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959; 31:426-428.
28.Rahimiyan Zarif B, Azin M, Amirmozafari N. Increasing the bioethanol yield in the presence of furfural via mutation of a native strain of Saccharomyces cerevisiae. Afr J Microbiol Res 2011; 5:651-656.
29.Aly MM, Tork S, Alakilli SY. Molecular characterization of chitiniolytic Bacillus pumilus isolated from marine habitats and enhancement of chitinase production by mutation. Univ scholar in Biotechnol 2011;1:14-21.
30.Shafique S, Bajwa R, Shafique SH. Mutation of Alternaria tenuissima FCBP-252 for hyper-active α-amylase. Indian J Exp Biol 2009; 47:592-596.
31.Azin M, Noroozi E. Random mutagenesis and use of 2-deoxy-D-glucose as an antimetabolite for selection of α-amylase-overproducing mutants of Aspergillus oryzae. World J Microbiol Biotechnol 2001; 17:747-750.
32.Li XH, Yang HJ, Roy B, Park EY, Jiang, LJ, Wang D. Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet. Microbiol Res 2009; 10:1-10.
33.Deshpande SK, Dhotmange MG, Chakrabarti T, Shastri PN. Production of cellulose and xylanase by Trichoderma reesei (QM 9414 mutant), Aspergillus niger and mixed culture by solid state fermentation (SSF) of water hyacinth (Eichhornia crassipes). Indian J Chem Technol 2008; 15:449-456.
34.Muhammad MJ, Haq I, Irfana M. Multistep mutagenesis for the overexpression of cellulase in Humicola insolens. Pak J Bot 2011; 43:669-677.
35.Poorna CA, Prema P. Production and partial characterization of endoxylanase by Bacillus pumilus using agro industrial residues. Biochem Eng J 2006; 33:106-112.
36.Oliveira LA. Production of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes. Bioresource Technol 2006; 97:862-867.
37.Battan B, Sharma J, Dhiman SS, Kuhad RC. Enhanced production of Cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry. Enzyme Microb Tech 2007; 41:733-739.
38.Knob A, Carmona C. Xylanase production by Penicillium sclerotiorum and its characterization. World Appl Sci J 2008; 4:277- 283.