Date Published: January 14, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Manju Sharma, Chhavi Mahajan, Manpreet S. Bhatti, Bhupinder Singh Chadha.
This study reports thermophilic fungus Malbranchea flava as a potent source of xylanase and xylan-debranching accessory enzymes. M. flava produced high levels of xylanase on sorghum straw containing solidified culture medium. The optimization of culture conditions for production of hemicellulases was carried out using one factor at a time approach and Box–Behnken design of experiments with casein (%), inoculum age (h) and inoculum level (ml) as process variables and xylanase, β-xylosidase, acetyl esterases and arabinofuranosidase as response variables. The results showed that casein concentration between 3.0 and 3.5 %, inoculum age (56–60 h) and inoculum level (2–2.5 ml) resulted in production of 16,978, 10.0, 67.7 and 3.8 (U/gds) of xylanase, β-xylosidase, acetyl esterase and α-l-arabinofuranosidase, respectively. Under optimized conditions M. flava produced eight functionally diverse xylanases with distinct substrate specificity against different xylan types. The peptide mass fingerprinting of 2-D gel electrophoresis resolved proteins indicated to the presence of cellobiose dehydrogenase and glycosyl hydrolases suggesting the potential of this strain in oxidative and classical cellulase-mediated hydrolysis of lignocellulosics. Addition of xylanase (300 U/g substrate) during saccharification (at 15 % substrate loading) of different pretreated (acid/alkali) substrates (cotton stalks, wheat straw, rice straw, carrot grass) by commercial cellulase (NS28066) resulted in 9–36 % increase in saccharification and subsequent fermentation to ethanol when compared to experiment with commercial enzyme only. High ethanol level 46 (g/l) was achieved with acid pretreated cotton stalk when M. flava xylanase was supplemented as compared to 39 (g/l) with xylanase without xylanase addition.
Hemicellulose is the second most abundant biopolymer in plant cell wall after cellulose which exists as O-acetyl-4-O-methylglucuronoxylan in hardwoods and as arabino-4-O-methylglucuronoxylan in softwoods, while xylan in grasses and annual plants are typically arabinoxylans consisting of a β-1,4-linked backbone of d-xylopyranosyl residues to which α-l-arabinofuranosyl (araf) residues are linked at C-3 and C-2 (Scheller and Ulvskov 2010). Owing to its complexity, the complete hydrolysis of xylan requires the action of main and side chain cleaving enzymes including endo-β-1, 4-xylanase (E.C. 184.108.40.206), β-D-xylosidase (E.C. 220.127.116.11), α-l-arabinofuranosidase (E.C.18.104.22.168), acetyl xylan esterase (E.C. 22.214.171.124) and ferulic or p-coumaric acid esterase (E.C.126.96.36.199) (Shallom and Shoham 2003). In recent years, xylanases have received a great deal of research attention particularly because of their biotechnological potential in food, feed, and pre-bleaching of pulps in paper industries. Each of these applications do require xylanases with distinct physico-chemical properties in terms of their mode of action, substrate specificity, pH, temperature optima, etc. (Polizeli et al. 2005). Recent reports on xylanases (derived from recombinant T. reesei Multifect®) suggest important role of these xylanases in enhancing the capability of the cellulases for hydrolysis of pretreated substrates (Hu et al. 2013). However, owing to increasing biotechnological importance of thermostable xylanases, there is a need to identify novel and catalytic efficient sources of xylanases for their role in improving these processes. Few of the important thermophilic fungal strains (Thermomyces lanuginosus, Myceliophthora sp., Melanocarpus albomyces, Paecilomyces thermophila) as sources of thermostable xylanases have been reported in recent past (Saraswat and Bisaria 1997; Sonia et al. 2005; Yang et al. 2006; Badhan et al. 2007). This study reports production of functionally diverse xylanases and xylan-debranching accessory enzymes by a novel thermophilic fungal strain M. flava isolated from composting soils (Sharma et al. 2008). The preliminary studies showed that culture produced appreciable levels of xylanase, however, for commercial application it is desirable to improve titres of xylanase and xylan-debranching accessory enzymes and, therefore, culture conditions for optimization of enzyme production using solid-state fermentation was studied. Furthermore, culture extracts were also used for profiling multiple xylanases, β xylosidases, α-l-arabinofuranosidases and acetyl esterases as well as secretome analysis using peptide mass fingerprinting approaches. The role of M. flava xylanases in enhancing saccharification of differently pretreated substrates for subsequent fermentation to ethanol was also examined.
Thermophilic fungus M. flava was found to produce high levels of xylanase under SSF using sorghum straw, a cheap and readily available carbon source. The xylanases from M. flava characterized as thermostable and catalytically efficient from this lab (Sharma et al. 2010) demonstrated the potential in improving bioconversion of lignocellulosics into ethanol. The culture is being further developed into commercially important source of xylanase through strain development approaches.