Mode of Action of a Family 75 Chitosanase from Streptomyces avermitilis

Abstract

Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).

Chitosans for delivery of nucleic acids

“…The enzymatic hydrolysis of chitin and chitosan has been proposed as an alternative method for the production of chitin/chitosan oligomers but at elevated cost. Chitosanases are enzymes that can depolymerize chitosan chains and many studies have been done to identify their mechanism of action [96][97][98][99]. The efficiency of the enzyme will depend on the distribution of A and D units along the polymer chain.…”

Reassessment of chitosanase substrate specificities and classification

“…All chitosanases can cleave D–D, while class I chitosanases, for example, from Streptomyces sp. N174 (belonging to GH 46), can additionally cleave A–D 16 – 20 ; class III chitosanases, like from Bacillus circulans MH-K1 (also belonging to GH 46), can, in addition to D–D, also cleave D–A 21 – 23 . Class II enzymes, such as the Bacillus sp.…”

“…Thus, this chitosanase generally seems to act more efficiently than other chitosanases on polymers containing more A-units. More recent studies have indicated that some enzymes cleave different patterns of acetylation with different efficiencies, such that the product composition depends, to some extent, on the substrate’s F A 20 , 28 . Furthermore, high substrate concentrations can result in substrate inhibition and reduce the cleavage efficiency 29 , 30 .…”

Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants

“…NMR has long been the method of choice, but requires comparatively large amounts of sample and gives only average values for DP and DA in COS mixtures, while DP and DA even of complex mixtures of COS can easily be determined using MS, where both matrixassisted laser desorption ionization and elecrospray ionization based methods have been employed (Tømmeraas et al, 2001;Trombotto et al, 2008). One advantage of NMR is that it easily gives information on the acetylation state of the reducing end unit, and to some extent also of the non-reducing and penultimate units (Heggset et al, 2012). However, Bahrke et al (2002) developed a method based on a reducing end tagging by using 2-aminoacridone, for sequencing of COS up to DP 12 using MS, and advanced MS-based sequencing methods are currently being developed (Haebel et al, 2007;Moerschbacher et al, unpublished).…”