Development of Mutants of Melanocarpus albomyces for Hyperproduction of Xylanase

다국어 초록 (Multilingual Abstract)

The wild type filamentous fungus, Melanocarpus albomyces, produces many commercially valuable enzymes, including Xylanases and Xylan-debranching enzymes with low activity. In this paper, we report for the first time the development of M. albomyces mutants from vegetative spores. Profuse sporulation of M. albomyces was induced on Potato Carrot Agar medium. These spores,when subjected to chemical mutation, led to the isolation of the hyper-xylanase producing mutant, viz, M. albomyces IITD3A. Various parameters including number of spores,nitrogen source and C/N ratio of the medium were optimized for production of xylanase by the mutant in a shake flask culture. Under controlled pH at 7.8, the mutant produced highly active xylanase with 415 IU/mL after 36h of growth on soluble alkaline lignocellulosic extract in a 14-L fermentor. The overall productivity of xylanase was 8-fold higher than the wild type culture with11,530 IU/L/h. The enzyme can be easily stored at 37oC for 50 days by addition of a small amount of the preservative - thiomersal.
Also, for long term storage, a lyophilized powder form of the enzyme can be used which retained 100% of its activity for > 50 days. When assayed at pH 7.5 and temperature 55oC, the xylanase retained 100% of its original activity,and also at pH 9.0, it retained > 50% of its activity for 2 h,which is promising for its application in the pulp and paper industry.

참고문헌 (Reference)

1 Polizeli, M. L. T. M, "Xylanases from fungi: Prop­erties and industrial applications" 67 : 577-591, 2005

2 Smith, D. C, "Xylanase production by Aspergillus awamori: Development of a medium and optimiza­tion of the fermentation parameters for the production of extra-cellular xylanase and β-xylosidase while maintaining low protease production" 38 : 883-890, 1991

3 Shatalov, A. A, "Xylanase pre-treatment of giant reed organosolv pulps: Direct bleaching effect and bleach boosting" 25 : 248-256, 2007

4 Smith, G, "Variations in inocula and their influence on the productivity of antibiotic fermentations" 2 : 261-266, 1980

5 Miller,G.L, "Use of dinitrosalicylic acid regent for deter­mination of reducing sugar" 31 : 426-428, 1959

6 Techapun, C, "Thermostable and alkaline-tolerant cellulase-free xylanase produced by thermotolerant Streptomyces sp. Ab106" 93 : 431-433, 2002

7 Qinnghe, C, "The screening of culture conditions and properties of xylanase by white-rot fungus Pleurotus ostreatus" 39 : 1561-1566, 2004

8 Righelato, R. C, "The influence of maintenance energy and growth rate on the meta­bolic activity, morphology and conidiation of Penicillium chrysogenum" 50 : 399-412, 1968

9 Morton,A.G, "The induction of sporulation in mould fungi" 153 : 548-569, 1961

10 Lichstein, H. C, "Studies of the effect of sodium azide on microbic growth and respiration I. The action of sodium azide on microbic growth" 47 : 221-230, 1944

11 Skromne, I, "Starvation stress modulates the expression of the Aspergillus nidulans brlA regu­latory gene" 141 : 21-28, 1995

12 Wulf, P. D, "Screening and mutational improvement of a D-ribose secreting Candida pelliculosa strain" 82 : 1-7, 1996

13 Magos,L, "Review on the toxicity of ethylmercury, includ­ing its presence as a preservative in biological and pharmaceuti­cal products" 21 : 1-5, 2001

14 Biely, P, "Remazol brilliant Blue-xylan: A soluble Chromogenic substrate for xylanases" 160 : 536-541, 1988

15 Kontkanen, H, "Purifi­cation and characterization of a novel steryl esterase from Mel­anocarpus albomyces" 39 : 265-273, 2006

16 Saraswat, V, "Purification, characteriza­tion and substrate specificities of xylanase isoenzymes from Mel­anocarpus albomyces IIS 68" 64 : 1173-1180, 2000

17 Kang, M. K, "Purification and characterization of two xylanases from alkalophilic Cephalospo­rium sp. strain RYM-202" 62 : 3480-3482, 1996

18 Khandeparkar, R, "Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112" 157 : 315-325, 2007

19 Kiiskinen, L. L, "Purification and characterization of a novel laccase from the ascomycete Melano­carpus albomyces" 59 : 198-204, 2002

20 Oliveira, L. A, "Pro­duction of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes" 97 : 862-867, 2006

21 Haltrich, D, "Production of fungal xylanases" 58 : 137-161, 1996

22 Panagiotou, G, "Production of cellulolytic and xylanolytic enzymes by Fusarium oxysporum grown on corn stover in solid state fermen­tation" 18 : 37-45, 2003

23 Dwivedi, P, "Parthenium sp. as a plant biomass for the production of alkali-tolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation" 33 : 581-588, 2009

24 Dobrev, G. T, "Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design" 98 : 2671-2678, 2007

25 Haltrich, D, "Optimization of a culture medium for increased xylanase production by a wild strain of Schizophyllum" 15 : 854-860, 1993

26 Papagianni, M, "Morphological develop­ment of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelia mor­phology" 5 : 3-, 2006

27 Kiiskinen, L. L, "Laccase from Melanocarpus albomyces binds effectively to cellulose" 576 : 251-255, 2004

28 Bailey, M. J, "Laboratory testing of methods for assay of xylanase activity" 23 : 257-270, 1992

29 Maheshwari, R, "Isolation and culture of a thermophilic fungus, Melanocarpus albomyces and factors influencing the production and activity of xylanase" 131 : 3017-3027, 1985

30 Li, Y, "Improvement of xylanase production by Penicillium oxalicum ZH-30 using response surface methodology" 40 : 1381-1388, 2007

31 Bisaria, V. S, "Enzymatic preb­leaching of Kraft pulps for paper manufacture" 27-31, 2002

32 Hirvonen, M, "Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: Mechanistic implications based on the free and cellobiose-bound forms" 329 : 403-410, 2003

33 Tatum, E. L, "Chemical induction of colonial paramorphs in Neurospora and Synceph­alastrum" 109 : 509-511, 1949

34 Sahai, V, "Cellulose-free nutrient medium for enhanced productivity and activity of cellu­lase-free enzymes"

35 Saraswat, V, "Biosynthesis of xylanolytic and xylan debranching enzymes in Melanocarpus albomyces IIS 68" 83 : 352-357, 1997

36 Song, A. R, "A study of the utilization of Pleurotus eryngii with different carbon and nitrogen sources" 8 : 10-14, 2001

37 Chandra, R. K, "A cellulase-free xyla­nase from alkali-tolerant Aspergillus fischeri Fxn1" 17 : 309-314, 1995