Enzymatic and molecular characterization of α-1,3-glucanase (AglST2) from <i>Streptomyces thermodiastaticus</i> HF3-3 and its relation with α-1,3-glucanase HF65 (AglST1)

Abstract

Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5-20% (w/v) NaCl, and 1 mM metal ions Mn and Co. On the other hand, it was inhibited by 1 mM of Ag, Cu, Fe and Ni. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0-7.0 at up to 60°C and in the presence of 0-20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.

Functional analysis of α-1,3-glucanase domain structure from <i>Streptomyces thermodiastaticus</i> HF3-3

“…1 in Section "Introduction", Agl-ST, which belongs to bacterial type GH87 family, has a domain architecture different from those of the known GH87 family enzymes. Furthermore, the previous report indicated that Agl-ST showed a high similarity to mycodextranase that specifically hydrolyzes α-1,4-glycosidic linkages in polysaccharide comprising α-D-glucose units alternatively linked (1 → 3) and (1 → 4) (Cherdvorapong et al, 2019). Given these results, to investigate the function of each domain of Agl-ST and their synergetic effect, and to compare them with those of the other α-1,3-glucanases and mycodextranases, is of great interest from the viewpoint of their evolution and their future applications.…”

“…Meanwhile, Agl-ST consists of four domains, namely, N-terminal β-SW, a catalytic domain, UC, and C-terminal DS. Figure 1 shows the domain architecture of Agl-ST made on the basis of the amino acid sequence reported previously (Cherdvorapong et al, 2019). Comparison of Agl-ST with the related enzymes revealed a high similarity to mycodextranase, whereas it had a low identity with the known α-1,3-glucanases.…”

“…In the previous studies, we have purified and characterized two types of α-1,3-glucanase (Agl-ST), Agl-ST (previously referred to as Agl-ST2) and the catalytic unit of Agl-ST (CatAgl-ST, previously referred to as Agl-ST1) from Streptomyces thermodiastaticus HF3-3, which are derived from the same gene. We clarified that Agl-ST could be categorized as a new group of α-1,3-glucanase (Cherdvorapong et al, 2019;Suyotha et al, 2017). Hakamada et al (2008) and Suyotha et al (2013) reported that GH 87 enzymes have a multi-domain structure containing α-1,3-glucan-binding modules.…”

Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α‐1,3‐glucanase from Paenibacillus glycanilyticus FH11

“…The primary structures and biochemical properties of the bacterial GH87 enzymes have already been investigated [8,[19][20][21][22][24][25][26][27][28][29][30]. However, the reaction mode, three-dimensional structure, substrate recognition, and catalytic mechanism of these GH87 enzymes had not been described in detail in previous studies.…”

“…Previously, we have isolated many a-1,3-glucanaseproducing bacteria from soil samples using mutan as an inducer of the enzymes and have characterized their GH87 a-1,3-glucanases, such as Agl-KA from Bacillus circulans strain KA-304 [24,25], Agl-FH1 and Agl-FH2 from P. glycanilyticus strain FH11 [21,26], and mycodextranase-type a-1,3-glucanase (Agl-ST1 and Agl-ST2) from Streptomyces thermodiastaticus strain HF3-3 [22,[27][28][29][30]. These GH87 a-1,3-glucanases specifically hydrolyzed only a-1,3-glucan and did not act on the other polysaccharides we tested, namely a-1,4-glucan (amylose), a-1,6-glucan (dextran), b-1,3-glucan (cardran), or b-1,4-glucan (cellulose) [18,21,22].…”