Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution

Abstract

The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

Cloning, Secretory Expression and Characterization of a Unique pH-Stable and Cold-Adapted Alginate Lyase

“…(Accession: QGU34249.1), and alginate lyase from Vibrio sp. (Accession: ASA33935.1) [21][22][23][24]. The results show that Alyw201 contains the typical conserved motifs of the PL7 family, such as "RT/SELRE," "QIH," and "MYFKAG" (Figure 4).…”

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger

“…T opt and t 1/2 are related but different parameters with the former indicating enzyme adaptivity to a higher temperature and the later, enzyme longevity at a certain temperature. Other CBM fusions also improved catalytic activities of the related enzymes (Furtado et al 2015;Khan et al 2013;Kittur et al 2003;Li et al 2015;Mai-Gisondi et al 2015;Mangala et al 2003;Ye et al 2011). 3; Table 1).…”

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties

“…Recent studies have revealed that NCRs within polysaccharide depolymerases are important elements that are inactive during substrate degradation but that affect enzymatic characteristics and oligosaccharide productivities (30,47). Among PL7 alginate lyases, the effects of most NCRs remain unclear, with the exception of the CBM13 module of AlyL2 of Agarivorans sp.…”

“…Among PL7 alginate lyases, the effects of most NCRs remain unclear, with the exception of the CBM13 module of AlyL2 of Agarivorans sp. strain L11 (30), although crystal structures and biochemical properties of many catalytic domains have been elucidated (25,48). Deletion of the CBM13 domain from AlyL2 changed its substrate preferences, and the presence of this module increased the disaccharide proportion of its products.…”

“…In contrast, the NCR fragments cannot degrade substrates but may affect the enzymes' properties. In this regard, the effects of NCRs on alginate lyases are relatively unknown, except for a CBM13 module (30).…”