Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

Abstract

We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40°C, where it showed a K m of 83 mg ml ؊1 , a k cat of 3.9 ؋ 10 2 h ؊1 , and a k cat /K m of 4.7 h mg ؊1 ml ؊1 on colloidal chitin. On chitobiose, the K m , k cat , and k cat /K m were 203 M, 1.3 ؋ 10 2 h ؊1 , and 0.62 h ؊1 M ؊1 , respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.

Transglycosylation by Chitinase D from Serratia proteamaculans Improved through Altered Substrate Interactions

“…But in this study there was no temporal difference between the hydrolytic and TG activities of SpChiD. As both hydrolytic and TG activities start simultaneously at 0 min (20), even at very low enzyme and substrate concentrations (supplemental Fig. S4) the mutational approach could be most appropriate for improving the TG activity.…”

“…Among the characterized single domain chitinases, SpChiD showed significant TG activity with a potential to synthesize CHOS with DP up to 13 (20). SpChiD therefore attained importance as a unique hyper-TG chitinase produced by an endophytic bacterium.…”

“…We have characterized a family 18 TG chitinase D from Serratia proteamaculans (SpChiD) that had a molecular mass of 44.4 kDa, producing TG products like DP7, DP10, DP11-12, and DP11-DP13 from DP3, DP4, DP5, and DP6 substrates, respectively (20). The SpChiD displayed TG activity only for 90 min with 2 mM chitotetraose (DP4) as substrate; later only hydrolytic products with lower DP were produced.…”

Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation

“…These features are common to processive, endoacting enzymes (64,65), and polysaccharide binding is observed in the grooves of both BcsZ and PgaB. In comparison, GH39 members, with the exception of PslG, exhibit exo-acting hydrolytic activity that arises as a direct consequence of an active site pocket that permits the correct orientation of the substrate to allow for hydrolysis of only the terminal sugar.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Synthesis by a hypertransglycosylating processive endochitinase of Serratia proteamaculans 568 (Purushotham & Podile, 2012) Chitosans TOF (DHB)…”