Isolation of β-1,3-Glucanase-Producing Microorganisms from Poria cocos Cultivation Soil via Molecular Biology

β-1,3-Glucanase is considered as a useful enzymatic tool for β-1,3-glucan degradation to produce (1→3)-linked β-glucan oligosaccharides with pharmacological activity properties. To validly isolate β-1,3-glucanase-producing microorganisms, the soil of Wolfiporia extensa, considered an environment rich in β-1,3-glucan-degrading microorganisms, was subjected to high throughput sequencing. The results demonstrated that the genera Streptomyces (1.90%) and Arthrobacter (0.78%) belonging to the order Actinomycetales (8.64%) in the phylum Actinobacteria (18.64%) were observed in soil for P. cocos cultivation (FTL1). Actinomycetes were considered as the candidates for isolation of glucan-degrading microorganisms. Out of 58 isolates, only 11 exhibited β-1,3-glucan-degrading activity. The isolate SYBCQL belonging to the genus Kitasatospora with β-1,3-glucan-degrading activity was found and reported for the first time and the isolate SYBC17 displayed the highest yield (1.02 U/mg) among the isolates. To check the β-1,3-glucanase contribution to β-1,3-glucan-degrading activity, two genes, 17-W and 17-Q, encoding β-1,3-glucanase in SYBC17 and one gene QLK1 in SYBCQL were cloned and expressed for verification at the molecular level. Our findings collectively showed that the isolates able to secrete β-1,3-glucanase could be obtained with the assistance of high-throughput sequencing and genes expression analysis. These methods provided technical support for isolating β-1,3-glucanase-producing microorganisms.

In bacteria, numerous recombinant and wild enzymes have been characterized from different sources, e.g., Streptomyces [8], Arthrobacter sp. [9,10], Cellulosimicrobium cellulans [11], Nocardiopsis sp. [12], Paenibacillus sp. [13], Thermotoga neapolitana [14], Bacillus circulan [15]. However, The classification results at the taxa phylum level are depicted in Figure 1. Proteobacteria (42.08%), Actinobacteria (18.64%), Acidobacteria (12.41%) and Bacteroidetes (5.95%) are considered as the dominant phyla as they contained over 5% of high-quality sequences in FLT 1 . The high-quality sequences not less than 1% were also classified into other subdominant phyla (Table S1). Eleven of abovementioned groups accounted for 96.13%. The remaining 16 phyla, in which effective sequences occurred at <1% abundance of the high-quality sequences, were defined as rare phyla in FLT 1 . Euryarchaeota and Thaumarchaeota belonging to the archaea domain accounted for 0.04% and 0.01% of the total high-quality sequences among FLT 1 , respectively (Table S1). The dominant phyla of FLT 1 and FLT 2 were compared. The composition of dominant phyla in FLT 2 was similar to those of FLT 1 . The phylum Actinobacteria in FLT 1 was over 5.06% of high-quality sequences those in FLT 2 . The abundances of other dominant phyla in FLT 2 were greater than those in FLT 1 . Root exudates containing various primary and secondary plant metabolites have distinct influences on insect herbivores, nematodes, and microbes underground, in addition to deterring competing plants [27]. Thus, the microbial communities in FTL 1 and FTL 2 might illustrate the relationship of the abundances of dominant phyla with its use of the main metabolites produced by P. cocos. The abundance of Actinobacteria is more likely to relate with β-1,3-glucan produced by P. cocos. It is reasonable to consider that bacteria belonging to the phylum Actinobacteria probably have ability to secrete β-1,3-glucanase for β-1,3-glucan degradation. Certainly, it cannot be excluded that other dominant phyla can found microorganisms with β-1,3-glucanase activity.
Molecules 2018, 23, x FOR PEER REVIEW 3 of 18 of the total high-quality sequences among FLT1, respectively (Table S1). The dominant phyla of FLT1 and FLT2 were compared. The composition of dominant phyla in FLT2 was similar to those of FLT1. The phylum Actinobacteria in FLT1 was over 5.06% of high-quality sequences those in FLT2. The abundances of other dominant phyla in FLT2 were greater than those in FLT1. Root exudates containing various primary and secondary plant metabolites have distinct influences on insect herbivores, nematodes, and microbes underground, in addition to deterring competing plants [27]. Thus，the microbial communities in FTL1 and FTL2 might illustrate the relationship of the abundances of dominant phyla with its use of the main metabolites produced by P. cocos. The abundance of Actinobacteria is more likely to relate with β-1,3-glucan produced by P. cocos. It is reasonable to consider that bacteria belonging to the phylum Actinobacteria probably have ability to secrete β-1,3glucanase for β-1,3-glucan degradation. Certainly, it cannot be excluded that other dominant phyla can found microorganisms with β-1,3-glucanase activity. The soil for P. cocos cultivation (FLT1) was used as the candidate sample for isolating β-1,3glucanase-producing microorganisms. To understand the diversity and structure of β-1,3-glucanaseproducing microorganisms in FLT1, a combination of literature research and data analysis among dominant phyla was conducted in detail ( Figure 2). Although Proteobacteria is the main dominant phylum that exists in FLT1, few reports refer to β-1,3-glucanase-producing microorganisms belonging to the phylum Proteobacteria from FLT1. Indeed, detailed data about microorganisms able to secrete glycoside hydrolases are available for this phylum, e.g., Sphingomonas [28,29], Sphingomonadaceae [30], Burkholderia [31], Pseudomonas [32]. The soil for P. cocos cultivation (FLT 1 ) was used as the candidate sample for isolating β-1,3-glucanase-producing microorganisms.
To understand the diversity and structure of β-1,3-glucanase-producing microorganisms in FLT 1 , a combination of literature research and data analysis among dominant phyla was conducted in detail ( Figure 2). Although Proteobacteria is the main dominant phylum that exists in FLT 1 , few reports refer to β-1,3-glucanase-producing microorganisms belonging to the phylum Proteobacteria from FLT 1 . Indeed, detailed data about microorganisms able to secrete glycoside hydrolases are available for this phylum, e.g., Sphingomonas [28,29], Sphingomonadaceae [30], Burkholderia [31], Pseudomonas [32]. Only the glycoside hydrolases selected from the genera Pseudomonas and Burkholderia that act on β-1,3-glucan have been exhaustively reviewed. The genus Burkholderia is worthless to isolate for its pathogenicity. Rare organisms able to degrade β-1,3-glucan were observed in the phyla Acidobacteria and Bacteroidetes from FLT 1 , except a characterized β-glucosidase from Mucilaginibacter sp. Strain QM49 [33]. The genera Arthrobacter and Streptomyces, accounting for 0.78% and 1.90% of the total high-quality sequences, were observed in the phylum Actinobacteria. It was noteworthy that both of the genera belong to the order Actinomycetales, which have been extensively reported as a source of β-1,3-glucanase [34][35][36]. FLT 1 widely harbored the order Actinomycetales (8.64%) when it was compared with others especially the genus Pseudomonas (1.76%) belonging to the order Pseudomonadales (3.44%). Moreover, the remarkable presence of Actinobacteria was found in FLT 1 but few that of microorganisms were observed in FTL 2 . A member of the order Actinomycetales is often called an actinomycete. It is well known that actinomycetes have unrivalled capacity to produce over two-thirds of natural antifungal metabolites [37]. Actinomycetes of the genus Streptomyces is well known as the largest genus of Actinobacteria, with properties of biological control. Overall, FLT 1 probably harbored a number of actinomycetes able to secrete β-1,3-glucanase. Actinomycetes can be classified into probiotics. Thus, the soil for P. cocos cultivation was used as the candidate sample for isolating β-1,3-glucanase-producing actinomycetes.

Identification of Glucan-Degrading Microorganisms
Colonies of actinomycetes were visible after the dilution of soil cultured on yeast casamino acids extract and dextrose agar (YCED). The isolates with β-1,3-glucan-degrading activity were screened among preferred actinomycetes. Out of 58 actinomycetes, only 11 among formed a clear halo around the colony after inoculation, indicating that they were able to degrade glucan ( Figure 3). A positively relation can be found between the size of clear halo and the enzyme activity. The size of clear halo in SYBC26 and SYBCQL were obviously smaller than others. Thus both of them are weak to degrade β-1,3-glucan. Only the glycoside hydrolases selected from the genera Pseudomonas and Burkholderia that act on β-1,3-glucan have been exhaustively reviewed. The genus Burkholderia is worthless to isolate for its pathogenicity. Rare organisms able to degrade β-1,3-glucan were observed in the phyla Acidobacteria and Bacteroidetes from FLT1, except a characterized β-glucosidase from Mucilaginibacter sp. Strain QM49 [33]. The genera Arthrobacter and Streptomyces, accounting for 0.78% and 1.90% of the total high-quality sequences, were observed in the phylum Actinobacteria. It was noteworthy that both of the genera belong to the order Actinomycetales, which have been extensively reported as a source of β-1,3-glucanase [34][35][36]. FLT1 widely harbored the order Actinomycetales (8.64%) when it was compared with others especially the genus Pseudomonas (1.76%) belonging to the order Pseudomonadales (3.44%). Moreover, the remarkable presence of Actinobacteria was found in FLT1 but few that of microorganisms were observed in FTL2. A member of the order Actinomycetales is often called an actinomycete. It is well known that actinomycetes have unrivalled capacity to produce over two-thirds of natural antifungal metabolites [37]. Actinomycetes of the genus Streptomyces is well known as the largest genus of Actinobacteria, with properties of biological control. Overall, FLT1 probably harbored a number of actinomycetes able to secrete β-1,3-glucanase. Actinomycetes can be classified into probiotics. Thus, the soil for P. cocos cultivation was used as the candidate sample for isolating β-1,3-glucanase-producing actinomycetes.

Identification of Glucan-Degrading Microorganisms
Colonies of actinomycetes were visible after the dilution of soil cultured on yeast casamino acids extract and dextrose agar (YCED). The isolates with β-1,3-glucan-degrading activity were screened among preferred actinomycetes. Out of 58 actinomycetes, only 11 among formed a clear halo around the colony after inoculation, indicating that they were able to degrade glucan ( Figure 3). A positively relation can be found between the size of clear halo and the enzyme activity. The size of clear halo in SYBC26 and SYBCQL were obviously smaller than others. Thus both of them are weak to degrade β-1,3-glucan. Ten isolates were identified to the genus Streptomyces, with the exception of SYBCQL belonging to the genus Kitasatospora, via 16S rRNA gene analysis. Each of the 16S rRNA gene sequences from isolates were aligned and submitted to the GenBank database, and all the sequences showed a high identity match (99%) to sequences obtained from the GenBank database (Table 2). Four isolates were closer to S. cellostaticus and S. capoamus. Two isolates showed high similarities to S. cinerochromogenes and S. coelescens. Three isolates were homologous to S. indiaensis, S. viridochromogenes, and K. phosalacinea, respectively, while another was homologous to S. olivogriseus and S. filipinensis. Phylogenetic analysis verified the taxonomic affiliations searched by BLAST alignment (Figure 4). Ten isolates were identified to the genus Streptomyces, with the exception of SYBCQL belonging to the genus Kitasatospora, via 16S rRNA gene analysis. Each of the 16S rRNA gene sequences from isolates were aligned and submitted to the GenBank database, and all the sequences showed a high identity match (99%) to sequences obtained from the GenBank database (Table 2). Four isolates were closer to S. cellostaticus and S. capoamus. Two isolates showed high similarities to S. cinerochromogenes and S. coelescens. Three isolates were homologous to S. indiaensis, S. viridochromogenes, and K. phosalacinea, respectively, while another was homologous to S. olivogriseus and S. filipinensis. Phylogenetic analysis verified the taxonomic affiliations searched by BLAST alignment (Figure 4). The genus Streptomyces as a main member of actinomycetes was abundant in FLT 1 based on high-throughput sequencing. The genus Streptomyces as a main member of actinomycetes was abundant in FLT1 based on highthroughput sequencing.   The results of identification were in agreement with the analysis of microbial communities at the genus level in FTL 1 . The genus Kitasatospora, is homologous to the genus Streptomyces, belonging to the order Actinomycetales among the phylum Actinobacteria [38]. Thus, SYBCQL able to degrade β-1,3-glucan was isolated from FLT 1 under the same conditions.

Enzyme Activity Assay
The isolates that formed a clear halo around the colony ( Figure 3) were determined to have the ability to degrade glucan in an exhausted culturing medium ( Table 3). The isolates able to degrade β-1,3-glucan were feeble, as compared with that of Streptomyces rutgersensis [39] and Streptomyces torulosus PCPOK-0324 [16]. SYBC17 showed the highest yield of glucan-degrading activity (1.02 U/mg) among all isolates obtained from actinomycetes. Although the specific activity of SYBCQL was lower than that of others, the genus Kitasatospora with β-1,3-glucan-degrading activity was found and reported for the first time. In general, β-glucosidases participate in β-1,3-glucan degradation along with β-1,3-glucanases. Thus SYBCQL and SYBC17 with β-1,3-glucanases activity for β-1,3-glucan degradation were further verify at the molecular level.

Gene Clone and Analysis
One gene encoding β-1,3-glucanase was amplified from the genomic DNA of SYBCQL and named QLK1, encoding the deduced protein QLK1. Based on the genomic DNA of SYBC17, two β-1,3-glucanase genes were found and named 17-W, and 17-Q. Both correspond to the deduced proteins 17-W and 17-Q. PCR products were checked by 1% agarose gel electrophoresis and sequenced after TA cloning.
The residues 1-37 of QLK1 and the residues 1-30 of 17-W was identified as N-terminal signal peptides, according to SignaIP analysis. The mature protein QLK1 consisted of 391 residues with a deduced molecular mass of 40.4 kDa. The mature protein 17-W contained 389 residues and its deduced molecular mass was the same as QLK1. Meanwhile, 17-Q without an N-terminus leader sequence encoded a mature protein with a deduced molecular mass of 48.1 kDa ( Figure 5). Each of the putative amino acid sequences has a catalytic domain similar to GH 16, based on align the protein sequences from GenBank database. QLK1 and 17-W, with a potential carbohydrate-binding domain (CBM), similarly belong to the regions of CBM 13 from Streptomyces at the C-terminus sequence. The functional domains of QLK1 and 17-W were found to be similar to β-1,3-glucanase from Streptomyces sp. S27 [35]. A glycine-rich region was observed between the functional domains of GH 16 and CBM 13 in QLK1 and 17-W. The region was also found in the linker structure in β-1,3-glucanase from Streptomyces sp. S27 [35] and S. sioyaensis [8]. The C-terminus domain of 17-Q was grouped into CBM family 6, found in several xylanases, rather than CBM family 13. The functional domains of 17-Q was similar to β-1,3-glucanase from S. sioyaensis [8].
functional domains of QLK1 and 17-W were found to be similar to β-1,3-glucanase from Streptomyces sp. S27 [35]. A glycine-rich region was observed between the functional domains of GH 16 and CBM 13 in QLK1 and 17-W. The region was also found in the linker structure in β-1,3-glucanase from Streptomyces sp. S27 [35] and S. sioyaensis [8]. The C-terminus domain of 17-Q was grouped into CBM family 6, found in several xylanases, rather than CBM family 13. The functional domains of 17-Q was similar to β-1,3-glucanase from S. sioyaensis [8]. The CBMs of QLK1 and 17-W exhibited a structure like that of the ricin B-chain classified in CBM 13 members. The ricin B lectin domain is composed of three homologous regions as the QXW (Gln-X-Trp) repeats ( Figure 6) [40]. There is a hypothesis that Gln works in substrate binding and Trp help to form the hydro phobic core [41]. The CBM of 17-Q is homogeneous to the CBMs belonging to family 6. Generally, members of CBM family 6 bind to xylan by connecting with the xylanase domain. For instance, the CBM of xylanase A from Clostridium stercorarium has been suggested to bind xylan and act as an important role in xylan hydrolysis [42]. Family 6 CBMs containing multiple distinct ligand binding sites present a unique ligand binding surface to recognize the non-reducing end of β- The CBMs of QLK1 and 17-W exhibited a structure like that of the ricin B-chain classified in CBM 13 members. The ricin B lectin domain is composed of three homologous regions as the QXW (Gln-X-Trp) repeats ( Figure 6) [40]. There is a hypothesis that Gln works in substrate binding and Trp help to form the hydro phobic core [41]. The CBM of 17-Q is homogeneous to the CBMs belonging to family 6. Generally, members of CBM family 6 bind to xylan by connecting with the xylanase domain. For instance, the CBM of xylanase A from Clostridium stercorarium has been suggested to bind xylan and act as an important role in xylan hydrolysis [42]. Family 6 CBMs containing multiple distinct ligand binding sites present a unique ligand binding surface to recognize the non-reducing end of β-1,3-linked-glucans [43]. The CBM of S. sioyaensis β-1,3-glucanase is probably considered as an extra ordinary CBM classified into family 6, based on its binding preference, especially due to its unwilling binding to xylan (Figure 7) [8]. The ligand binding sites of 17-Q are similar to those found in the CBM of S. sioyaensis β-1,3-glucanase and probably have the same binding preference. GH family 16 [44,45]. A Met residue was observed in the catalytic motif of endo-β-1,3-glucanases but not in endo-β-1,3-1,4-glucanases [11]. As shown in Figure 5, a specific consensus motif with putative catalytic residues is found among these β-1,3-glucanases. BglF is completely inactive when the mutants of the deduced catalytic residues Glu123Gln and Glu128Gln are created [12]. Thus, the putative catalytic residues are crucial among these hydrolases. Besides, Glu128 protonates the glycosidic oxygen of the scissile bond by acting as a general acid [44].  GH family 16 [44,45]. A Met residue was observed in the catalytic motif of endo-β-1,3-glucanases but not in endo-β-1,3-1,4-glucanases [11]. As shown in Figure 5, a specific consensus motif with putative catalytic residues is found among these β-1,3-glucanases. BglF is completely inactive when the mutants of the deduced catalytic residues Glu123Gln and Glu128Gln are created [12]. Thus, the putative catalytic residues are crucial among these hydrolases. Besides, Glu128 protonates the glycosidic oxygen of the scissile bond by acting as a general acid [44].  A highly consensus catalytic center for the hydrolysis of glycosidic bonds has been observed in GH family 16 [44,45]. A Met residue was observed in the catalytic motif of endo-β-1,3-glucanases but not in endo-β-1,3-1,4-glucanases [11]. As shown in Figure 5, a specific consensus motif with putative catalytic residues is found among these β-1,3-glucanases. BglF is completely inactive when the mutants of the deduced catalytic residues Glu123Gln and Glu128Gln are created [12]. Thus, the putative catalytic residues are crucial among these hydrolases. Besides, Glu128 protonates the glycosidic oxygen of the scissile bond by acting as a general acid [44].

Expression and Purification of Recombinant Enzymes
Recombinant enzymes were expressed with IPTG (isopropyl-β-D-1-thiogalacto-pyranoside) induction in E. coli BL21 (DE3) cells. Crude extracts in soluble form were purified using Ni + affinity chromatography and desalting chromatography was used to remove the excess salt. SDS-PAGE analysis confirmed that QLK1, 17-W and 17-Q were overexpressed successfully, with high purity of the enzymes. The presence of molecular masses was close to the theoretical masses according to the deduced amino acid sequences of the enzymes (Figure 9). The purified recombinants QLK1, 17-W and 17-Q had specific activities of 65.82 U/mg, 132.90 U/mg, and 14.70 U/mg, respectively (Table 5). 17-Q showed the highest yield among all purified recombinant enzymes. All the recombinant enzymes displayed a several times higher level of β-1,3-glucanase activity than wild-type. These results suggested that SYBCQL and SYBC17 with β-1,3-glucanases activity were successfully confirmed in molecular level.

Expression and Purification of Recombinant Enzymes
Recombinant enzymes were expressed with IPTG (isopropyl-β-D-1-thiogalacto-pyranoside) induction in E. coli BL21 (DE3) cells. Crude extracts in soluble form were purified using Ni + affinity chromatography and desalting chromatography was used to remove the excess salt. SDS-PAGE analysis confirmed that QLK1, 17-W and 17-Q were overexpressed successfully, with high purity of the enzymes. The presence of molecular masses was close to the theoretical masses according to the deduced amino acid sequences of the enzymes (Figure 9). The purified recombinants QLK1, 17-W and 17-Q had specific activities of 65.82 U/mg, 132.90 U/mg, and 14.70 U/mg, respectively (Table 5). 17-Q showed the highest yield among all purified recombinant enzymes. All the recombinant enzymes displayed a several times higher level of β-1,3-glucanase activity than wild-type. These results suggested that SYBCQL and SYBC17 with β-1,3-glucanases activity were successfully confirmed in molecular level.

Materials
SYBCQL and SYBC17 were used as genetic DNA sources. E. coli DH5α and E. coli BL21 (DE3) were purchased from TaKaRa (Dalian, China) and used as hosts for genes cloning and expression. The plasmid pUC19 and pCold II vector were bought from TaKaRa and used for constructing recombinant plasmid. Luria-Bertani (LB) medium with 50 µg/mL ampicillin was used in recombinant plasmid amplification. The genomic DNA extraction kit, LA Taq DNA polymerase with GC buffer, PCR clean-up kit and other DNA-modifying enzymes were bought from TaKaRa. Laminarin with an average BR of 98% was purchased from Shanghai Yuanye Bio-Technology Company (Shanghai, China). The powder of fruiting bodies of the Basidiomycete P. cocos was provided by Johncan International Company (Hangzhou, China). High-throughput sequencing was performed by Shanghai Shenggong Company (Shanghai, China). Other chemicals were all of analytical grade and commercially available.

High-Throughput Sequencing
To find the candidate isolates in FLT 1 , for screening β-1,3-glucanase-producing microorganisms, the microbial communities in the soil for P. cocos cultivation (FLT 1 ) and bulk soil (FLT 2 ) were investigated and compared.
Soil sampling was carried out in March 2017. The sphagnum and duff layers of the sampling area were removed, and P. cocos were found around the roots of pine trees using a soil knife. FLT 1 was collected from the soil around P. cocos growing in a township (28 • 35 N, 185 • 95 E), Liu'an City, Anhui Province, China. The bulk soil sample (FLT 2 ) was gathered approximately 2 m away from FLT 1 and just under the root zone of any grasses growing on the surface (pH 7.0). The soil of the 10 cm depth layer was collected using an auger with a diameter. To remove stones and roots, both of the soil samples were timely sieved (2 mm mesh) in the field. The treated samples were then kept under a low temperature maintained by ice until molecular analysis.
High-throughput sequencing in molecular analysis and data processing were conducted as described previously [46]. After DNA extraction, PCR amplification, and pyrosequencing, the MiSeq-generated raw sequences were submitted to the DDBJ database (accession number: DRA006753). The raw MiSeq-generated sequences were further processed using the soft-ware Prinseq (PRINSEQ-lite 0.19.5) [47] and the software package of Mothur1.30 with "pre.cluster" command [48]. The available sequences were clustered into operational taxonomic units (OTUs) and the thresthod value of sequences similarity was set at 0.97. Based on the results of OTU clustering, the most abundant sequence as the representative sequence of OTU was acquired and subjected to various types of analysis.
Taxonomic assignment was accomplished by the Ribosomal Database Project (RDP) Classifier according to Bergey's taxonomy [49]. A bootstrap cutoff of 80% was used to assign the obtained sequences to each taxonomy levels. The evolutionary relationships and abundance of the dominant phyla in FLT 1 at the genus level were visualized using the ete3 (Environment for Tree Exploration) package in python.

Isolation and Identification of Glucan-Degrading Microorganisms
Actinomycetes were screened from FLT 1 by serial dilution and spread-plate techniques [50]. FLT 1 (5 g) was mixed with 100 mL sterile distilled water and diluted to 10 −5 . One hundred microliter of the different dilutions were grown on the plates containing 0.03% yeast extract, 0.03% casamino acid, 0.03% D-glucose, 0.05% K 2 HPO 4 , and 1.8% agar (w/v) in triplicates, respectively. Cyclohexamide (100 µg/mL) was added to resist fungal contamination after autoclaving. The plates were incubated at 28 • C for 1-2 weeks. Typical actinomycetes colonies were picked out according to morphological characteristics as well as microscopic examination. The morphologically distinct colonies were then purified on the original media at 28 • C for one week, stored at 4 • C. Furthermore, glucan-degrading actinomycetes were inoculated on agar plates containing the powder of fruiting bodies of Basidiomycete P. cocos (0.5%, w/v) and aniline blue (0.005%, w/v), and then formed a clear halo around the colony [51].
To identify the unknown isolates, the selected isolates were incubated in 3 mL ISP-2 medium under rotary shaking at 30 • C for 48 h. A volume of 1.5 mL culture was centrifuged at 8000× g for 1 min. The pellet was then washed once with distilled water and used to extract genomic DNA. The genomes of each isolate were extracted following by the operating instruction of genomic DNA extraction kit (www.tiandz.com). 16S rRNA gene identification of glucanase-producing bacteria were amplified by using the universal primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ) and sent to Shanghai Shengong (Shanghai, China) for sequencing. A homology search of the closest phylogenetic neighbors was conducted using the online tool BLAST.

Determination of the Enzyme Activity
The isolates were inoculated aerobically with rotary shaking at 200 rpm and 30 • C for 72 h in a medium (per liter) containing laminarin 5 g, K 2 HPO 4 1 g, NaNO 3 3 g, KCl 0.5 g, MgSO 4 ·7H 2 O 0.5 g, and FeSO 4 ·7H 2 O 0.5 g. The cultures were centrifuged at 10,000× g at 4 • C and the culture filtrates were harvested for activity assay. The standard activity assay for β-1,3-glucan degradation was obtained by measuring the formation of reducing sugar using a colorimetric method [16]. Culture filtrates of the strains (500 µL) were mixed with 500 µL of 0.5% (w/v) laminarin in 100 mM sodium acetate buffer (pH 5.5). The reaction was conducted at 50 • C for 60 min and terminated by heating for 5 min at 100 • C. Then 2 mL of 1% dinitrosalicylate (DNS) was added into the reaction solution and the mixture was boiled for 10 min. The mixture was placed in an ice bath and then measured at 540 nm using the spectrophotometer. According to the standard assay conditions, one unit (U) of the activity was defined as the amount of enzymes that can liberate 1 µmol of glucose in one minute. All experiments were set to repeat, with triplicates of each treatment. Protein concentrations were measured by the method of Bradford [52] using bovine serum albumin as a standard.

Cloning and Expression of β-1,3-Glucanase Genes
A genome analysis from the NCBI database was conducted, with the genomic DNA of Kitasatospora setae KM-6054 and Streptomyces griseochromogenes ATCC 14511 employed as the templates and synthetic primers ( Table 6). The genome of SYBCQL and SYBC17 has been extracted by genomic DNA extraction kit. The plasmid pCold II DNA and the vector pUC19 DNA was isolated using a plasmid miniprep kit. Then the coding sequences were amplified by the polymerase chain reaction (PCR) using LA Taq DNA polymerase with GC buffer, and sequenced after TA cloning. The nucleotide sequences were deposited in the GenBank database (accession number: 17-W, MH190407; 17-Q, MH190408; QLK1, MH190409.). Subsequently PCR products were ligated with the pcold II vector after both were digested with Hind III and Xba I. The recombinant plasmid was then transformed into E. coli BL21 (DE3) competent cells. Transformants containing the recombinant enzymes were picked from the single colony and inoculated overnight at 37 • C in ampicillin-supplemented LB. Moreover, the overnight cultured transformants (1 mL) were transferred into 50 mL of fresh LB medium with the addition of 100 µg/mL ampicillin and grown at 37 • C to a cell density of 0.6~0.8. To induce the expression of the recombinant enzymes, IPTG was then added to a final concentration of 0.4 mM and the cultivation continued for 24 h at 15 • C. Cells were harvested by centrifugation at 4 • C and 8000× g for 10 min, and resuspended in 100 mM sodium acetate buffer at pH 5.5. Cells were lysed by sonication for 15 min on ice, and cell supernatants was collected by centrifugation (8000× g, 10 min at 4 • C) for further purification.

Purification of the β-1,3-Glucanases
To purify the recombinant proteins with six histidine residues, an AKTA Avant system at 6 • C was used (GE Healthcare, Uppsala, Sweden), followed by desalting with a HisTrap TM column (GE Healthcare, Uppsala, Sweden). The cell supernatant (crude enzyme) was applied to a HisTrap HP column (GE Healthcare) equilibrated with binding buffer A (100 mM sodium acetate, 5 mM imidazole, 500 mM NaCl, pH 5.5), and was eluted with buffer B (100 mM sodium acetate, 500 mmol/L imidazole, and 500 mmol/L NaCl, pH 5.5) using an imidazole step gradient of 0% to 100% buffer B. The collected fractions with β-1,3-glucanase activity were assayed using the former described method. The purified proteins were loaded on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of purified proteins was tested by the method described above.

Conclusions
In the present paper, actinomycetes with the ability to degrade β-1,3-glucan were isolated using high-throughput sequencing combined with culture-dependent techniques. Both strains SYBCQL and SYBC17 able to secrete β-1,3-glucanase for β-1,3-glucan degradation were verified at the molecular level. It was suggested that these methods could be applied to effectively isolate β-1,3-glucanase-producing microorganisms, which is useful for the screening of other metabolite-producing microorganisms from specific environment.

Supplementary Materials:
The following are available online. Figure S1: Rarefaction curves based on the OTUs at the cutoff of 97% 16S rRNA sequence similarity, Table S1: Relative abundances (% of total good-quality sequences) of all phyla in each soil sample. The dominant phyla are marked in shade (>1% of good quality sequences in at least one sample), and the total abundances in each soil sample are displayed at the bottom of the latter two lines. Table S2: The abundance of taxa genus levels in soil samples. The taxa represented within the top 30 abundances at the taxa genus levels and beyond the top 30 abundances at the taxa genus levels are classified into other levels.
Author Contributions: Q.W. (Qiulan Wu) performed most of the experiments and wrote the paper. Z.G. and Y.C. provided intellectual input to the study design. X.D. and Q.W. (Qi Wang) revised the manuscript. X.L. provided all financial support for this research project.
Funding: This research was funded by the Collaborative Innovation Involving Production, Teaching and Research Funds of Jiangsu Province (BY2014023-28) and the Agricultural Support Project, Wuxi Science and Technology Development (CLE01N1310).