Characterization of Stackebrandtia nassauensis GH 20 Beta-Hexosaminidase, a Versatile Biocatalyst for Chitobiose Degradation

Abstract

An unstudied β-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The β-N-acetylhexosaminidase was able to hydrolyze β1,2-linked, β1,3-linked, β1,4-linked, and β1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the β-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.

Genomic and transcriptomic landscapes and evolutionary dynamics of molluscan glycoside hydrolase families with implications for algae-feeding biology

“…Approximately 20% members of all molluscan GH family genes contain one or more CBMs, which is higher than the rough ratio (7%) of the CAZy database entries containing one or more CBMs [4] . It is interesting that most of the CBM-containing families (GH9, GH10, GH18, GH20) that participate in cellulose, hemicellulose and chitin digestion are expanded in molluscs [30] , [74] , [75] , [76] . Especially in the GH18 family, 35% of copies possessed up to five CBMs located at the N/C-terminus or interspersed among catalytic domains, which can promote the association of the enzyme with the substrate.…”

Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell

“…To determine the biochemical parameters of MxChi, a colorimetric assay based on the derivatization of free amine with o-phthalaldehyde (OPA) was used, a method reported previously by Lv et al [29,32]. The assay design is based on the coupling of the chitinase, activity beta-hexosaminidase, and GlcNAc deacetylase [32], which catalyzes the formation of chitobiose from chitin, the formation of GlcNAc from chitobiose, and the formation of GlcN from GlcNAc, respectively (Figure S3).…”

“…To determine the biochemical parameters of MxChi, a colorimetric assay based on the derivatization of free amine with o-phthalaldehyde (OPA) was used, a method reported previously by Lv et al [29,32]. The assay design is based on the coupling of the chitinase, activity beta-hexosaminidase, and GlcNAc deacetylase [32], which catalyzes the formation of chitobiose from chitin, the formation of GlcNAc from chitobiose, and the formation of GlcN from GlcNAc, respectively (Figure S3). Briefly, typical chitinase activity reaction mixture was quenched by heating to remove the chitinase, then β-N-acetylhexosaminidase (SnHex) was added for overnight to fully convert the product of chitinase to GlcNAc, GlcNAc deacetylase CmCBDA was used at last.…”

“…Chitinases have found diverse industrial, pharmaceutical, and agricultural applications, including chitin rich wastes degradation [43], diseases [20], biocontrol against phytopathogenic fungi and insects [4], and so on, which made scientists particularly interested in discovering new chitinases, while a satisfactory recombinant form with the robust chitinase properties is difficult to obtain. In this study, high-throughput manner by microplate-reader in a coupled enzymatic assay was used to explore the properties of Mx-Chi, which was based on the reported assay by Wang et al and Lv et al (Figure S3) [29,32]. The purified MxChi was most active at pH 7.0 in phosphate buffer (Figure 3a), and it was stable within pH 4.0-8.0 as it retained more than 70% of its maximal activity, indicating that the enzyme is a neutral chitinase.…”

“…In this report, we wanted to confirm that MxChi can be used in an enzymatic cascade, in which GlcN can be produced enzymatically from chitin. Firstly, GlcNAc was generated by adding MxChi and SnHex [32] to the same reaction mixture containing colloidal chitin (Figure 5a), and the efficiency of MxChi and SnHex were successfully evaluated in a time-course reaction (Figure 5b). Only GlcNAc was detected after 12 h compared with the standard GlcNAc on TLC.…”