In
the present study, we carried out a comprehensive investigation
of glycoside hydrolase (GH) 46 model-chitosanases based on cleavage
specificity classification to understand their unknown bifunctional
activity. We for the first time show that GH46 chitosanase CsnMHK1
from Bacillus circulans MH-K1, which
was previously thought to be strictly exclusive to chitosan, can hydrolyze
both chito- and cello-substrates. We determined the digestion direction
of bifunctional chitosanase CsnMHK1 from class III and compared it
with class II chitosanase belonging to GH8, providing insight into
unique substrate specificities and a new perspective on its reclassification.
The results lead us to challenge the current understanding of chitosanase
substrate specificity based on GH taxonomy classification and suggest
that the prevalence from the common bifunctional activity may have
occurred. Altogether, these data contribute to the understanding of
chitosanase recognition and hydrolysis toward chito- and cello-substrates,
which is valuable for future studies on chitosanases.
Endo-chitosanases (EC 3.2.1.132) are generally considered
to selectively
release functional chito-oligosaccharides (COSs) with degrees of polymerization
(DPs) ≥ 2. Although numerous endo-chitosanases have been characterized,
the digestion specificity of endo-chitosanases needs to be further
explored. In this study, a GH46 endo-chitosanase OUC-CsnPa was cloned,
expressed, and characterized from Paenibacillus sp.
1–18. The digestion pattern analysis indicated that OUC-CsnPa
could produce monosaccharides from chitotetraose [(GlcN)4], the smallest recognized substrate, in a random endo-acting manner.
Especially, the enzyme specificities during chitosan digestion including
the regulation of product abundance through a transglycosylation reaction
were also evaluated. It was hypothesized that an insertion region
in OUC-CsnPa may form a strong force to be involved in stabilizing
(GlcN)4 at its negative subsite for efficient hydrolysis.
This is the first comprehensive report to reveal the digestion specificity
and subsite specificity of monosaccharide production by endo-chitosanases.
Overall, OUC-CsnPa described here highlights the previously unknown
digestion properties of the endo-acting chitosanases and provides
a unique example of possible structure–function relationships.
Chitooligosaccharides (COSs) and D-glucosamine (GlcN), the most valuable biomolecules due to their various physiological functions, can be available inexpensively from chitosan by bio-enzymatic degradation, in which chitosanases play a key role. On the other hand, pretreatment of chitosan is the necessary procedure in the bioproduction by enzymatic hydrolysis. Therefore, the green and efficient pretreatment strategy as well as superior enzyme activity are the most critical aspects in scaled up production. This study investigated environmentally friendly and efficient enzymatic hydrolysis of powdery chitosan (PC) pretreated by ball-milling. First, heterologous expression yielded OUC-CsnPT, a glycoside hydrolase family 46 chitosanase with a predominant specific activity of 5346.56 U/mg at pH 6.0°C and 45°C. The endo-type chitosanase OUC-CsnPT hydrolyzed chitosan to produce GlcN and chitobiose [(GlcN) 2 ]. Then, ball-milling was employed to pretreat PC to promote chitosanase OUC-CsnPT hydrolysis, and the effectiveness was characterized by scanning electron microscope, X-ray diffraction, and FTIR-attenuated total reflection. Through the development of the novel route, the yield of COSs and GlcN for ball-milling powdery chitosan increased by 10.8-folds with a concentration of 14.5 μmol/ml. Hence, this technique presents a promising strategy suited to be a straightforward and environmentally friendly option for oligosaccharides production, broadening the theoretical basis for PC biodegradation.
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