The recombinant AbfB displayed molecular and physical characteristics similar to most α-L-arabinofuranosidases belonging to the glycosyl hydrolase (GH) family 54. The molecular mass (MW) of 67 kDa of the recombinant AbfB estimated on the 10% SDS-PAGE (Fig. 4.8a) was similar to that obtained by Veen et al. (1991), which falls within the range of molecular masses (49-70 kDa) described by other workers (Rombouts et al., 1988; De Ioannes et al., 2000; Koseki et al., 2000; Fritz et al., 2008; De Wet et al., 2008). However, the MW of 67 kDa observed on SDS-PAGE was greater than the MW of 52.4 kDa calculated from the gene sequence using the DNAMAN computer software. The difference between the observed and calculated AbfB molecular masses could in part be attributed to possible post translational modification such as glycosylation, which in this case could be estimated to be 29%. The glycosylation is required for the enzyme to attain full activity and stability upon secretion (Gweyn, 1992; Conesa et al., 2001).
The recombinant AbfB can be utilised optimally at a broader temperature range (40- 55ºC) (Fig. 4.7a) and under a wide range of acidic conditions (pH 3.0-pH 6.0) (Fig. 4.7b). The molecular and physical characteristics of the AbfB are in agreement with properties reported for α-L-arabinofuranosidases belonging to glycoside hydrolase (GH) family 54 http://afmb.cnrs-mrs.fr/CAZY/GH_54.htm (De Ioannes et al., 2000; Kaneko et al., 2006; De Wet et al., 2008). Therefore, the recombinant AbfB has characteristics of a robust and flexible enzyme technology for selective xylan hydrolysis. The results showed that the recombinant AbfB was stored in its crude form without detectable loss of activity for over 24 months at 4ºC. Furthermore, from
100 the 4ºC storage temperature, the enzyme was stable for more than 144 h (6 days) at 26-37ºC (Fig. 4.11a) and during hydrolysis of xylans performed at 40ºC for 16-24 h at pH 5.0 (Fig. 4.11b). Therefore, it was possible to recycle more than 95% of the recombinant AbfB activity against pNPA after catalysing release of arabinose from polymeric xylan substrates (Fig. 4.11b). The observed effect is typical of enzymes that do not stay bound to their substrates after the catalytic reactions (Van Beilen and Li, 2002). However, the activity of the recombinant AbfB was reduced by over 50% at temperatures of > 60ºC. A loss of over 95% of the activity was observed within five minutes of incubation at 80ºC (Fig 4.7d). Therefore, recombinant AbfB temperature and pH stability suggest that direct application of the enzyme in lignocelluloses processing such as the kraft pulp and paper making processes would require some adjustment of the temperature and pH. However, the stability in storage and recyclability after xylan hydrolysis of the recombinant AbfB would particularly reduce the cost of production and handling. Furthermore, the recyclability of the enzyme after xylan hydrolysis implies less contamination of the enzymes of the xylan product. Therefore, the recombinant AbfB presents a robust and flexible technology that can be used for selective xylan hydrolysis and be compatible for use in processes where high purity standards are required.
4.3.4 AbfB kinetics and substrate specificity
The recombinant AbfB, produced both in the standard and CCSL enriched media displayed Michaelis-Menten (saturation) kinetic properties that reached equilibrium at 40 mM pNPA. The saturation curves provided estimated Km values between 6-10 mM upon catalysis of pNPA of varying concentration (Fig. 4.9). The observed Km values are within the values reported by Tajana et al. (1992) who reported Km values of 10- 12.5 mM for two α-L-arabinofuranosidases obtained from Streptomyces diastaticus. However, these values are higher for Km values of 4.8 x 10-4 M reported for fungal arabinofuranosidases from A. niger by Rombouts et al. (1988). Although the Km value is an intrinsic parameter that should remain constant for a specific enzyme (Shuler and Kargi, 2002), the results from this study showed that the Km values were different for the same AbfB when produced in 2xMM medium and in 2xMM medium enriched with 2% CCSL (Fig. 4.9). This observation is in agreement with Shuler and Kargi (2002) who indicated that similar enzymes are likely to have different Km values (thus possessing different affinity levels to the same substrate) if subjected to different
101 temperature or pH conditions and when secreted by different organisms. A low Km suggests that the enzyme has high affinity towards the substrate and the value corresponds to the substrate concentration that would give 50% of the maximal forward velocity (Vmax) (Shuler and Kargi, 2002). Therefore, the Km values obtained for the AbfB suggests lower affinity towards the pNPA compared to the corresponding AbfB reported by Rombouts et al. (1988).
The AbfB presents a potential technology for selective removal of arabinose side chains from a broader spectrum of lignocellulosic materials without degrading the main xylan chains, which confirmed that the recombinant AbfB lacked β-xylanase activity. The recombinant AbfB catalysed liberation of α-1,2- and α-1,3-bonded arabinose side chains from low viscosity wheat xylan and corn fibre xylan substituted with arabinose and MeGlcA side chains (Fig. 4.10c). In addition, the AbfB was capable of removing 7% of α-1,3-bound arabinose from larchwood arabinogalactan and α-1,5-bonded arabinose from debranched arabinan (Fig. 4.10a). Such characteristic is an indication of the broad substrate specificity of the GH family 54 AbfBs (De Ioannes et al., 2000). The ability of the AbfB to remove α-1,5-bonded arabinose in this study is also in agreement with studies by Luonteri et al. (1998) whereby 5% of available arabinose was released from linear beet α-1,5-bound arabinose, but contradicts the results on lack of action by the recombinant AbfB from
A. pullulans on similar substrate reported by De Wet et al. (2008). Therefore, it
appears substrate specificity of the AbfB may have changed. However, the removal of the arabinose from the low viscosity wheat arabinoxylan and corn fibre xylan caused visible precipitation (Fig. 4.10b), indicating the ability of recombinant AbfB for use in insolubilising water soluble xylans. Therefore, the recombinant AbfB has potential use for production of insoluble xylan hydrogels from xylans.
The AbfB had greater affinity towards arabinoxylans compared to arabinoglucuronoxylans as such higher degree of arabinose removal was obtained from oat spelt than from arabinoglucuronoxylans extracted from bagasse, bamboo and
P. patula (Chapter 3) and from H2O2 bleached bagasse xylans. The results have shown that 20% of available arabinose was released from oat spelt arabinoxylan compared to 9% released from arabinoglucuronoxylans extracted from bagasse by the Hoije method (Fig. 3.10c). The relatively limited release of the arabinose from
102 substrates such as arabinoglucuronoxylans of bagasse and P. patula origin could be attributed to limited accessibility of the AbfB to the side chains due to steric hindrance from α-D-MeGlcA side chains. Therefore, synergistic application of the recombinant AbfB and of pure α-D-glucuronidase (with polymeric substrate specificity and xylanase free) might improve the release of arabinose.
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4.4
CONCLUSION
An Aspergillus niger D15[abfB] strain selectively secreted α-L- arabinofuranosidase (AbfB) with improved purity and crude enzyme specific activity of 2.9 U mg-1 under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and glucoamylase terminator (glaAT). The AbfB secretion was growth
associated and as such the biomass growth and protein yield per unit biomass were improved by cultivating the recombinant A. niger in 2xMM media enriched with 2% concentrated corn steep liquor. The AbfB was stable under cultivation, storage and application conditions at temperatures between 30-60ºC and pH 3.0-6.0 providing the possibility of multiple applications from a single AbfB dosage. The secreted recombinant α-L-arabinofuranosidase (AbfB) displayed polymeric xylan substrate specificity, thus having the ability to remove arabinose side chains from polymeric xylans and cause the xylans to precipitate. The extracellular overexpression of the recombinant AbfB in relatively purer and larger quantities than wild type strain, presents a novel biological tool for diversifying functional properties of xylans from various feedstocks as novel speciality additives, coatings and hydrogels encapsulation matrices.
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