J. Gen. Appl. Microbiol. Doi 10.2323/Jgam.2020.07.003 ©2021 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 1

Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2020.07.003 ©2021 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 1 2 Full Paper 3 Functional analysis of α-1,3-glucanase domain structure from Streptomyces thermodiastaticus 4 HF3-3 5 (Received May 25, 2020; Accepted July 31, 2020; J-STAGE Advance publication date: February 12, 2021) 6 7 Niphawan Panti1, Vipavee Cherdvorapong1, Takafumi Itoh2, Takao Hibi2, Wassana Suyotha3, 8 Shigekazu Yano4, and Mamoru Wakayama1* 9 1 10 Department of Biotechnology, Faculty of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 11 525-8577, Japan 2 12 Department of Bioscience and Biotechnology, Faculty of Bioscience and Biotechnology, Fukui 13 Prefectural University, Eiheiji, Fukui, 910-1195, Japan 3 14 Biotechnology for Bioresource Utilization Laboratory, Department of Industrial Biotechnology, 15 Faculty of Agro-industry, Prince of Songkla University, Hat Yai 90112, Thailand 4 16 Department of Biochemical Engineering, Graduate School of Sciences and Engineering, 17 Yamagata University, Jonan, Yonezawa, Yamagata 992-8510, Japan 18 19 Running head: Domain of Streptomyces α-1,3-glucanase 20 21 *To whom correspondence should be addressed. Tel: +81-77-561-2768 22 Fax: +81-77-561-2659; E-mail: [email protected] 1 23 24 Keyword: α-1,3-glucanase; α-1,3-glucan-binding activity; domain function; mycodextranase; 25 Streptomyces thermodiastaticus 26 27 Summary 28 α-1,3-Glucanase from Streptomyces thermodiastaticus HF3-3 (Agl-ST) has been classified in the 29 glycoside hydrolase (GH) family 87. Agl-ST is a multi-modular domain consisting of an N- 30 terminal β-sandwich domain (β-SW), a catalytic domain, an uncharacterized domain (UC), and a 31 C-terminal discoidin domain (DS). Although Agl-ST did not hydrolyze α-1,4-glycosidic bonds, 32 its amino acid sequence is more similar to GH87 mycodextranase than to α-1,3-glucanase. It 33 might be categorized into a new subfamily of GH87. In this study, we investigated the function 34 of the domains. Several fusion proteins of domains with green fluorescence protein (GFP) were 35 constructed to clarify the function of each domain. The results showed that β-SW and DS 36 domains played a role in binding α-1,3-glucan and enhancing the hydrolysis of α-1,3-glucan. 37 The binding domains, β-SW and DS, also showed binding activity toward xylan, although it was 38 lower than that for α-1,3-glucan. The combination of β-SW and DS domains demonstrated high 39 binding and hydrolysis activities of Agl-ST toward α-1,3-glucan, whereas the catalytic domain 40 showed only a catalytic function. The binding domains also achieved effective binding and 41 hydrolysis of α-1,3-glucan in the cell wall complex of Schizophyllum commune. 42 43 44 45 2 46 Introduction 47 α-1,3-Glucan, a water-insoluble linear α-1,3-linked homopolymer of glucose, is the main 48 component of extracellular polysaccharides synthesized from sucrose from Streptococcus mutans 49 and S. sobrinus by glycosyltransferases (GTFs) (GTF-I and GTF-SI) (Krzyściak et al., 2014; 50 Marotta et al., 2002). GTF-I synthesizes mostly insoluble glucan with an α-1,3-glycosidic bond, 51 and GTF-SI synthesizes a mixture of insoluble glucans with an α-1,3-glycosidic bond and an α- 52 1,6-glycosidic bond. GTF-I and GTF-SI have been considered to be the most important GTFs, 53 which are involved in the production of the major component of dental plaque in humans. Such 54 insoluble glucans facilitate the accumulation of carcinogenic bacteria, and other bacteria and, 55 consequently, enhance biofilm formation on dental surfaces, which causes dental caries, 56 gingivitis, and periodontitis that, in turn, lead to cardiovascular disease, rheumatoid arthritis, and 57 osteoporosis (Dervis, 2005; Griffin et al., 2009; Kinane et al., 2017; Pleszczyńska et al., 2015; 58 Wiater et al., 2013). 59 α-1,3-Glucan has been found in both ascomycetous and basidiomycetous fungi (Sipiczki et al., 60 2014). In Aspergillus nidulans, α-1,3-glucan was found during vegetative growth and shown to 61 play an important role in sexual development as an endogenous carbon source (Yoshimi et al., 62 2017; Zonneveld, 1972). In Schizosaccharomyces pombe, α-1,3-glucan has roles in secondary 63 septum formation and primary septum robustness during cell separation (Grün et al., 2005; 64 Hochstenbach et al., 1998; Suyotha et al., 2016). Moreover, α-1,3-glucan is related to the 65 virulent factors of pathogenic yeast, for example, in Cryptococcus neoformans, which is 66 pathogenic in humans. In the fungus Magnarporthe oryzae (Bacon et al., 1968; Reese et al., 67 2007), a pathogen causing rice blast, α-1,3-glucan is used during invasion not only to protect the 3 68 fungal cell wall from degradative enzymes secreted by the host but also to conceal chitin to delay 69 the innate immune response in plants (Fujikawa et al., 2012). 70 α-1,3-Glucanase is an enzyme that hydrolyzes the α-1,3-glycosidic bond of α-1,3-glucan, and 71 has been studied for the removal of dental plaque and as a biological control agent of pathogenic 72 fungi (Suyotha et al., 2016). This enzyme is classified into two types, fungal and bacterial, which 73 are referred to as the glycoside hydrolase (GH) families 71 and 87, respectively. 74 In the previous studies, we have purified and characterized two types of α-1,3-glucanase (Agl- 75 ST), Agl-ST (previously referred to as Agl-ST2) and the catalytic unit of Agl-ST (CatAgl-ST, 76 previously referred to as Agl-ST1) from Streptomyces thermodiastaticus HF3-3, which are 77 derived from the same gene. We clarified that Agl-ST could be categorized as a new group of α- 78 1,3-glucanase (Cherdvorapong et al., 2018; Suyotha et al., 2017). Hakamada et al. (2008) and 79 Suyotha et al. (2013) reported that GH 87 enzymes have a multi-domain structure containing α- 80 1,3-glucan-binding modules. For example, α-1,3-glucanase from Bacillus circulans KA-304 81 (Agl-KA) consists of N-terminal discoidin domain I (DS1), carbohydrate-binding module 6 82 (CBM6), threonine and proline repeats, discoidin domain II (DS2), uncharacterized domain (UC), 83 and C-terminal catalytic domain. Meanwhile, Agl-ST consists of four domains, namely, N- 84 terminal β-SW, a catalytic domain, UC, and C-terminal DS. Figure 1 shows the domain 85 architecture of Agl-ST made on the basis of the amino acid sequence reported previously 86 (Cherdvorapong et al., 2018). Comparison of Agl-ST with the related enzymes revealed a high 87 similarity to mycodextranase, whereas it had a low identity with the known α-1,3-glucanases. 88 However, Agl-ST is classified into α-1,3-glucanase based on its properties. 4 89 In this study, we constructed a fusion protein of putative domain with green fluorescence 90 protein (GFP) described in Fig. 2 and evaluated the function of each domain of Agl-ST from S. 91 thermodiastaticus HF3-3 by glucan binding and hydrolysis assays. 92 93 Materials and Methods 94 Microorganisms and culture conditions. Escherichia coli XL10-gold was used as a host for 95 the construction of recombinant plasmids containing a DNA fragment of domain GFP-fusion 96 proteins. It was cultivated in 5 ml of Luria Bertani (LB) broth (0.5% yeast extract, 1% 97 hipolypepton, and 1% NaCl (pH 7.0)) containing 50 µg/ml ampicillin at 37°C for 18 h. E. coli 98 Rosetta-gami B (DE3), which was used for the expression of proteins, was cultivated at 37°C for 99 18 h in 5 ml of LB broth (pH 7.0) containing 50 µg/ml ampicillin, 34 µg/ml chloramphenicol, 100 and 20 µg/ml kanamycin. S. commune IFO 4928 was grown at 30°C for 4 days in 5 ml of 101 Medium C containing 2% glucose, 1% hipolypepton, 0.3% yeast extract, 0.3% K2HPO4, and 5 102 mg/l thiamine, pH 7.0. For production on a large scale, the mycelial pellet was blended with a 103 blender at high speed for 1 min (Waring no. 7009), after which it was transferred to 250 mL of 104 Medium C and incubated with shaking at 100 rpm and 30°C for 4 days. The pellet was collected 105 by centrifugation at 13,000 rpm for 20 min, after which it was washed with distilled water three 106 times before lyophilization. The dried pellet was collected and ground as a powder, after which it 107 was used as a substrate for analyzing α-1,3-glucanase activity. 108 109 Preparation of GFP fusion proteins. The GFP gene was prepared from the pQBI-25fN1 plasmid 110 as a template, as described previously (Suyotha et al., 2013). The recombinant plasmids were 111 designed as pET-β-SW/Cat/UC/DS-GFP, pET-β-SW/Cat/UC-GFP, pET-Cat/UC/DS-GFP, pET- 5 112 β-SW-GFP, pET-Cat-GFP, pET-UC-GFP, and pET-DS-GFP (Fig. 1). The PCR fragments were 113 amplified by using the primer pairs NdeIβ-SW/BamDS, NdeIβ-SW/BamUC, NdeICat/BamDS, 114 NdeIβ-SW/Bamβ-SW, NdeICat/BamCat, NdeIUC/BamUC, and NdeIDS/BamDS, respectively 115 (Table 1). PCR was performed in a reaction mixture of KOD-Plus-Neo kit (Toyobo, Japan) with 116 the following condition for thermal cycling: 94°C for 2 min, followed by 35 cycles at 98°C for 117 20 s and 68°C for 2 min. Then, PCR products were purified using the Fast GeneTM Plasmid mini 118 kit (Genetics, Japan).

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