Xylooligosaccharides (XOS) are emerging prebiotics, widely used in food, medicine
and health care. For example, XOS selectively stimulates the growth and activity of
intestinal probiotic bacteria, and hence it is considered as safe functional food additives in
fortified food products. XOS is also a highly desirable prebiotic due to the small effective
dosage required which allows it easily to be incorporated into both functional foods and
dietary supplements at a competitive cost. XOS production practices can be broadly divided
into thermochemical and enzymatic methods. The latter is in general preferred over the
former due to its high specificity and absence of formation of non‐toxic byproducts. The
enzyme, endoxylanase, cleaves xylan polymer to XOS. In this study, endo-1,4-β-D-xylanase
(XynC) of B. subtilis KCX006 is cloned, overexpressed, and purified. The XOS was
produced from beechwood xylan and crude xylans isolated from sugarcane bagasse and
sorghum biomass using the purified recombinant xylanase. The XOS produced from these
xylans supported the growth of probiotic bacteria indicating their prebiotic activity. Further,
immobilization of xylanase, onto conventional organic polymers, improves enzyme‐stability
and efficacy of XOS production through its repeated use. To improve further the
performance, the purified recombinant xylanase was immobilized on ordered mesoporous as
well as nanosized materials, e.g., carbon (CMK-3 & AC), silica (SBA-15 & f-SiO 2 ) and
zirconia (ZMF-127 & n-ZrO 2 ).
The xylanase immobilized onto f-SiO 2 and n-ZrO 2 matrices produced higher
proportions of X2-X6 as compared with the free-xylanase. The xylanase immobilized on AC

and f-SiO2 showed only < 5% loss in the yield of XOS for eight repeated batch reactions. All
three nanomaterials supported the recycling of xylanase to produce XOS from xylan. The
adsorption of xylanase on nanomaterials do not shown any significant influence on optimum
pH for catalytic activity, however, the xylanase immobilized nanomaterials indicate a broad
optimum temperature range (50-65˚C). Further, the xylanase adsorption increases with the
increase of temperature (ca. 20 to 60˚C) for both carbon and zirconia nanoparticles, while for
f-SiO 2 increase in temperature (ca. 60°C) decreases the adsorption capacity. The adsorption
of xylanase on nanomaterials follow pseudo-second order kinetic model. The activation
energy for the xylanase adsorption corresponds to the physical adsorption range for carbon
and zirconia and the chemical adsorption range for f-SiO 2 . The adsorption of xylanase on
nanomaterials fitted well to the Freundlich isotherm model, and that the negative values of
free energy change indicate the adsorption is favorable and spontaneous. The maximum RA
of immobilized xylanase was found to be 93% for n-ZrO 2 . Owing to lower substrate
diffusion and higher steric hindrance, K m of immobilized xylanase increases.
Likewise, the adsorption of xylanase onto OMM reached equilibrium in 60 min and,
as before, the adsorption increases with the increase of temperature (ca. 20 to 60˚C). The
xylanase adsorption onto OMM also follows a pseudo-second order kinetics, and that the
thermodynamic parameters indicate spontaneous adsorption. However, the isotherm of the
xylanase adsorption on OMM differed from the adsorption of nanomaterials as the former
follows a typical Langmuir model, and that the maximum RA is 84%. Importantly, the
optimal pH and temperature conditions are not altered by immobilization unlike the
immobilized xylanase onto nanomatrices or other reported matrices reported in literature. In
addition, the Km values of the xylanase and that a higher level of X2-X6 was produced for
the xylanase immobilized onto SBA-15 and ZMF-127 due to high enzyme-loading. Besides,
the immobilized xylanase can be recycled up to eight repeated batches with retention of
activity up to 93% of the initial activity.
Publications:
1. Shivudu, G., Khan, S., Chandraraj, K. and Selvam, P. (2019). Immobilization of
recombinant endo-1,4-β-xylanase on ordered mesoporous matrices for xylooligosaccharides
production, ChemistrySelect, 4 (38), 11214-11221. DOI: 10.1002/slct.201901593
2. Shivudu, G., Chandraraj, K. and Selvam, P. (2020). “Production of xylooligosaccharides
from xylan catalyzed by endo-1,4-β-D-xylanase-immobilized nanoscale carbon, silica and
zirconia matrices, Molecular Catalysis, 484, 110745. DOI: 10.1016/j.mcat.2019.110745