Date Published: September 14, 2012
Author(s): Ziqing Gao, Duong Van Hop, Le Thi Hoang Yen, Katsuhiko Ando, Shuichi Hiyamuta, Ryuichiro Kondo.
Fusarium proliferatum NBRC109045 is a filamentous fungus isolated from Vietnamese forest due to high production of β-glucosidases. Production of the enzyme was studied on varied carbon source based mediums. The highest activity was obtained in medium containing 1% corn stover + 1% wheat bran (3.31 ± 0.14 U/ml). It is interesting to note that glucose (0.69 ± 0.02 U/ml) gave higher activity and just followed by cellobiose among the di- and mono-saccharides, which is generally regarded as a universal repressor of hydrolases. We improved the zymogram method to prove that in response to various carbon sources, F. proliferatum could express various β-glucosidases. One of the β-glucosidases produced by F. proliferatum growing in corn stover + wheat bran based medium was partially purified and proved to have high catalytic ability.
Biofuels derived from lignocellulosic biomass are emerging as promising alternatives to fossil fuels to meet the increasing global energy demands (Ragauskas et al. 2006). One of the key steps in bioconversion process is the enzymatic hydrolysis of the cellulose polymers in the biomass to monomeric sugars that are subsequently fermented to ethanol (Percival et al. 2006; Adsul et al. 2007). The three main categories of players in cellulose hydrolysis are cellobiohydrolases (or exo-1, 4-β-glucanases) (EC 126.96.36.199), endo-1, 4-β-glucanases (EC 188.8.131.52), and β-glucosidases (EC 184.108.40.206) (Beguin and Aubert 1994). The endo-1, 4-β-glucanases randomly attack cellulose in amorphous zones and release oligomers. The cellobiohydrolases liberate cellobiose from reducing and non-reducing ends. And finally β-glucosidases hydrolyze the cellobiose and in some cases the cellooligosaccharides to glucose (Ryu 1980; Wood 1985). Cellulose polymers are degraded to glucose through sequential and cooperative actions of these enzymes. Cellobiohydrolases and endoglucanases are often inhibited by cellobiose, making β-glucosidases important in terms of avoiding decreased hydrolysis rates of cellulose over time due to cellobiose accumulation (Workman and Day 1982). Low efficiency and high costs associated with the enzymatic hydrolysis process present a major bottleneck in the production of ethanol from lignocellulosic feedstocks (Banerjee et al. 2010). For the enzymatic conversion of biomass to fermentative sugar on a commercial scale, it is necessary to have all cellulolytic components at the optimal level. Since β-glucosidases activity is low in many microbial preparations used usually for the saccharification process (Enari 1983). It is necessary to supply additional β-glucosidases to such reaction. In order to optimize the use of different biomasses, it is important to identify new β-glucosidases with improved abilities on the specific biomasses as well as with improved abilities such as stability and high conversion rates. β-Glucosidases have potential roles in various fields such as the food, pharmacology and cosmetic industries and also in the valorisation of some products, due to the properties of this enzyme to convert and to synthesize biomolecules of high added value (Esen 1993). There are hundreds of different β-glucosidic flavor precursors in plants, and their hydrolysis often enhances the quality of the beverages and foods produced from them (Gϋnata 2003; Esen 2003). Aside from flavor enhancement, foods, feeds, and beverages may be improved nutritionally by release of vitamins, antioxidants, and other beneficial compounds from their glycosides (Opassiri et al. 2004). Indeed, β-glucosidase can either degrade or synthesize small carbohydrate polymers, depending on particular experimental conditions (Crout and Vic 1998). The β-glucosidases can be arranged in three groups related to localization: intracellular, cell wall associated, and extracellular. Primarily the extracellular β-glucosidases are of industrial interest (Soewnsen 2010). The number of fungal species on earth is estimated to 1.5 million of which as little as approximately 5% are known (Hawksworth 1991; 2001). So there is a statement that calls for all-out effort to unravel the potential of unknown species found in nature. The identification and characterization of new fungal species are often encountered in literature. Cuc Phuong Park and Ba Be Park is the old national one in Vietnam and boasts an engaging cultural and wildlife heritage and enchanting scenery. Covered in a dense forest, these landscapes are rich and diverse tropical and subtropical species of microorganisms for wood and plant degradation. In the present study, a potential β-glucosidases-producing fungus NBRC109045 was isolated from Ba Be national park and identified as Fusarium proliferatum. Under optimized conditions, F. proliferatum produces β-glucosidases with an activity of 3.3 U/ml based on pNPG as substrate and an activity of 426 U/ml based on cellobiose as substrate. In this paper, we described ways that (a) isolating and screening microbes to produce considerable quantities of β-glucosidases; (b) modifying the method of zymogram to prove that different carbon sources direct varied β-glucosidases expression in F. proliferatum; (c) assaying partial purification to prove high catalytic efficiency of β-glucosidase produced by F. proliferatum growing in corn stover + wheat bran based medium.
Cellobiose was considered as an inducer of cellulase which includes β-glucosidases (Mandels and Reese 1957). However, the amount of β-glucosidases when F. proliferatum grew in cellobiose based medium was less than that in corn stover + wheat bran based medium. When compared the yield of β-glucosidases in cellobiose based medium with that in corn stover + wheat bran based medium, F. proliferatum grew in cellobiose faster than that in corn stover + wheat bran (data not shown). This proved that cellobiose is an excellent growth substance for and is rapidly consumed, whereas corn stover + wheat bran is a relatively poor growth substance and is slowly consumed. The same phenomenon was observed by (Mandels and Reese 1960). They held the opinion that the inhibitory effect of cellobiose on β-glucosidases production seems to be related to rapid metabolism of the cellobiose.
The authors declare that they have no competing interests.