Actinomadura amylolytica YIM 77502^T (i.e., DSM 45822^T and CCTCC AA 2012024^T) was stored in our laboratory (Jiao et al., 2015). The E. coli DH5α strain (Invitrogen, United States) was used for cloning and the E. coli BL21 DE3 strain (Invitrogen, United States) was utilized as the host for expression of proteins. The pET28a vector (Invitrogen, United States) was used to construct recombinant plasmids. A. amylolytica YIM 77502^T was cultured on R2A medium (0.5 g/l yeast extract, 0.5 g/l peptone, 0.5 g/l glucose, 0.5 g/l soluble starch, 0.5 g/l casein acid hydrolysates, 0.5 g/l sodium pyruvate, 0.3 g/l K2HPO4, and 0.024 g/l MgSO4) at 40°C. Recombinant E. coli strains were grown at 37°C in lysogeny broth (LB) medium (1% tryptone, 0.5% yeast extract, and 1% sodium chloride; pH 7.0) containing kanamycin (50 μg/ml).

Actinomadura amylolytica YIM 77502^T was cultured in R2A and then genomic DNA was extracted using a MasterPure Gram-positive DNA Purification kit (Epicentre MGP04100) following the standard DNA isolation procedure recommended by the manufacturer with modifications (Wu et al., 2009). Whole-genome sequencing was performed on the Illumina HiSeq 2500-PE125 platform with massively parallel sequencing Illumina technology at the Beijing Novogene Bioinformatics Technology Co., Ltd. All high-quality paired reads were assembled using the SOAP de novo² into a number of scaffolds (Gu et al., 2013). Glimmer v3.0 (Chen et al., 2011) was used for gene prediction in assembled sequences of A. amylolytica YIM 77502^T. The sequence data described here have been deposited in GenBank (Accession number: CP032402). Proteins encoded by A. amylolytica were annotated using cluster of orthologous groups (COG) protein functional classification (Tatusov et al., 2003). Carbohydrate-active enzymes (CAZymes) of A. amylolytica YIM 77502^T were identified using the CAZyme Analysis Toolkit³ (Petit et al., 2015). The glycoside hydrolase families were analyzed using HMMER software based on the Pfam database⁴ (Finn et al., 2011).

Degradation of filter paper was assessed by inoculating strain A. amylolytica YIM 77502^T into a test tube containing filter paper basic medium (NaH₂PO₄, KNO₃, and MgSO₄) after static culture at 40°C for 1 month. A. amylolytica was cultured in cellulose basic medium (NaH₂PO₄, KNO₃, MgSO₄, and Avicel 2 g/l) at 40°C with shaking at 180 rpm for 14 days. The supernatant was harvested by centrifuging for 10 min at 12,000 × g at 4°C.

The cellulose degradation activity of the above fermentation broth was measured using CMC (Sigma, United States), Avicel (Sigma, United States), and cellobiose (Sigma, United States). Fermentation broth (100 μl) was added to 400 μl PBS buffer (pH 7) containing 1% (w/v) CMC or Avicel to test cellulase activity. This mixture was incubated at different temperatures (25–90°C) for 1 h. The release of reducing sugar from CMC was determined using the 3,5-dinitrosalicylic acid assay and measuring the absorbance at 540 nm (Miller, 1959). One unit (IU) of CMCase activity was defined as the volume of fermentation broth required to release 1 μmol of reducing sugar from CMC per minute. Hydrolytic products of the fermentation broth from CMC were identified by thin-layer chromatography (TLC) using silica gel 60 plates (Merck, Darmstadt, Germany) developed with 1-butanol/acetic acid/water (2:1:1, v/v/v). Sugars were detected by heat treatment at 120°C for 10 min after spraying the plates with freshly prepared 5% (v/v) H₂SO₄ in ethanol (Yin et al., 2015). Glucose (G1), cellobiose (G2), cellotriose (G3), and cellotetrose (G4) were used as sugar standards. The sugar standards was purchased from Sigma, United States.

The β-glucosidase activity of the fermentation broth was measured using cellobiose. Fermentation broth (100 μl) was added to 400 μl PBS buffer (pH 7) containing 1% (w/v) cellobiose at different temperatures (25–80°C) for 30 min to test β-glucosidase activity. The release of glucose from cellobiose was determined based on the absorbance at 490 nm of the mixture using a Glucose Oxidase Assay Kit (Abnova, China). One unit (IU) of β-glucosidase activity was defined as the volume of fermentation broth required to release 2 μmol glucose from cellobiose per minute.

The above fermentation broth was transferred into 5 kD ultra-filter concentrators (Sartorius, Germany) and centrifuged for 30 min at 4°C in a swing bucket rotor, resulting in the medium become concentrated by 30-fold. Extracellular protein was precipitated overnight by ammonium sulfate precipitation. The precipitate was resuspended in 150 μL supernatant. Proteins were isolated and digested as described by Hornburg et al. (2014). Peptides were desalted on C18 StageTips as previously described and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis (Schwanhäusser et al., 2013). The peptides were then separated using a nano high-performance liquid chromatography (HPLC; Thermo Fisher Scientific) with a C18 column. HPLC was directly coupled to a quadrupole-Orbitrap mass spectrometer via a nano electrospray ion source (Q Exactive^TM, Thermo Fisher Scientific). The mass spectrometry (MS) raw data was processed by MaxQuant (v. 1.3.8.2; Cox and Mann, 2008). MS/MS spectra were searched against the A. amylolytica YIM 77502^T genomic database.

The putative β-glucosidase genes aabgl1 (1335 bp) and aabgl2 (1431 bp) from the genomic sequences of A. amylolytica were amplified via polymerase chain reaction (PCR) with the four designed primers listed in Table 1 using TransStarFastPfu Fly DNA Polymerase (TransGen Biotech, China). The PCR cycle consisted of denaturation at 98°C for 3 min, followed by 34 cycles at 98°C for 10 s, 65°C for 20 s, and 72°C for 45 s, and then a final incubation at 72°C for 5 min for the final extension. The PCR products were cloned into the pET28a plasmid, which had been digested with BamH I and Hind III, using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China). Recombinant plasmids with the correct size fragments were sequenced. Subsequently, the DNA from the correct recombinant plasmids were transformed into E. coli BL21 (DE3) for protein expression.

DNA and protein sequences were aligned using the BLASTx and BLASTp programs (Madden, 2002)⁵, respectively. Signal peptides were predicted using SignalP (Bendtsen et al., 2004)⁶. The primary structures of the amino acid sequences were deduced and analyzed using EXPASY tools⁷. Multiple alignments with protein sequences of the closely related AaBGL1 and AaBGL2 (retrieved from NCBI database) were carried out using Clustal X (Thompson et al., 1997). Phylogenetic analyses were performed using the MEGA 5 software package (Tamura et al., 2011). Trees were constructed using the maximum likelihood (ML) method with a Poisson correction model. Structural models of AaBGL1 and AaBGL2 were generated with the MODELLER package (Sali and Blundell, 1993) using the β-glucosidase BglM-G1 mutant H75R (PDB ID, 5NS7; sequence identity, 47%) from a marine metagenome and β-xylosidase (PDB ID, 1GNX; sequence identity, 52%) from Streptomyces sp. as templates.

To express the recombinant AaBGL1 and AaBGL2 proteins, the above transformants were cultured overnight in LB medium containing 100 μg/ml kanamycin at 37°C and with shaking at 220 rpm. One ml of overnight culture was inoculated into 100 ml fresh LB medium containing 100 μg/ml kanamycin and incubated at 37°C with shaking at 220 rpm. To induce expression of the recombinant β-glucosidases, 0.1 ml of 100 mM IPTG was added to the cell suspension until the cells reached mid-exponential phase (OD₆₀₀≈0.6) and incubated at 25°C with shaking at 220 rpm for 6 h. Cell pellets were harvested by centrifuging at 8,000 × g at 4°C for 10 min and resuspended in 20 ml buffer A (20 mM sodium phosphate, 300 mM NaCl, and 10 mM Tris; pH 8.0).

The resuspended cells were disrupted by ultrasonication. The lysates were centrifuged at 12000 × g for 30 min at 4°C. Cell-free extracts were transferred onto a Ni-chelating affinity column (GE, United States) as the recombinant proteins possessed an N-terminal His-tag. The column was then washed with ten column volumes of buffer A and then ten column volumes of buffer A containing 20 mM imidazole (pH 8.0) and eluted with buffer A containing 300 mM imidazole (pH 8.0). The eluted proteins were desalted using disposable PD-10 Desalting Columns (GE, United States) and the desalted proteins were used for enzyme characterization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 10% polyacrylamide gel that was then stained with Coomassie brilliant blue dye R-250 (Liu et al., 2010). Protein concentrations were determined with Bradford Protein Assay Kit (Order NO. C503031, Sangon Biotech, China) using bovine serum albumin as the standard.

The kinetic constants (Vmax and Km) of AaBGL1 and AaBGL2 were determined using different concentrations of cellobiose (0.2–2 mg/ml) and pNPG (0.5–5.0 mg/ml) at the optimal pH and temperature for 5 min. The Vmax and Km of the β-glucosidases for cellobiose and pNPG were calculated using Lineweaver-Burk plots.

Enzymatic hydrolysis of different lignocellulosic materials, such as CMC, Avicel, birch sawdust, and corncob, was performed by adding the appropriate cellulases (Sangon Biotech, China) and/or AaBGL1 and AaBGL2 to a 0.5 ml reaction system containing 50 mM Na₂HPO₄-citric acid buffer (pH 6.0) and 0.5 ml of 1% (w/v) substrate at 40 and 50°C for 24 h. The dosages of cellulases and β-glucosidases (AaBGL1 and AaBGL2) were 0.2 and 0.02 U/mg, respectively, based on the dry weight of substrate (Ye et al., 2017). Pretreatment of birch sawdust and corncob with ionic liquid (1-ethyl-3-methylimidazolium acetate) was carried out according to the method described in previous studies (Cheng et al., 2012). After enzymatic hydrolysis, the reactions were terminated by boiling for 10 min. The supernatants were collected by centrifugation at 4°C at 10000 × g for 10 min. The glucose contents of the hydrolysis liquors were measured using the Glucose Oxidase Assay Kit (Abnova, China).

The complete genome of A. amylolytica YIM 77502^T consisted of a single circular 6.8 Mbp chromosome, and a G+C content of 73.28%. The genome contained 6,373 coding sequences with an average length of 922 bp. The general genomic features of A. amylolytica are listed in Supplementary Table S1. Among the predicted genes, 95.8% (6,105 genes) were assigned a function and 4.2% (268 genes) had unknown functions. In addition, 86.4% of the sequence encoded genes, while 13.6% belonged to internal sequences. The functions of these genes were mainly gene transcription (590), signal transduction (373), amino acid transport and metabolism (354), carbohydrate transport and metabolism (344), lipid transport and metabolism (324), energy production and conversion (289), coenzyme transport and metabolism (284), and cell wall/membrane/envelope biogenesis (273), as shown in Supplementary Figure S1.

A CAZyme analysis was conducted to identify potential enzymes with plant cell-wall degradation ability. Through this analysis, 81 carbohydrate-binding modules (CBMs) distributed in 12 families, 63 glycoside hydrolases (GHs) in 32 families, 2 polysaccharide lyases (PLs) in 2 families, 22 carbohydrate esterases (CEs) in 5 families, 71 glycosyl transferases (GTs) in 14 families, and 12 auxiliary activities (AAs) in 4 families were found to be encoded in the genome of A. amylolytica (Figure 1A). GH1 (glycoside hydrolase family 1) and GH3 were predicted to function as β-glucosidases and GH5, GH6, GH12, GH48 and GH74 were predicted cellulases. Altogether, 11 β-glucosidase genes and 9 cellulase genes were predicted in the genome of A. amylolytica (Figure 1B).

As shown in Figure 2A, filter paper was degraded by A. amylolytica YIM 77502^T after culturing 1 month. TLC demonstrated the hydrolytic products of fermentation broth from CMC were mainly glucose and cellobiose (Figure 2B). Fermentation broths from cultures in microcrystalline cellulose basic medium for 2 weeks exhibited CMC and cellobiose activity under test conditions. High CMCase activity was found from 50 to 65°C, where the highest β-glucosidase activity was observed at 50°C (Figures 2C,D).

The secretome of A. amylolytica was examined by MS. Secretome analysis revealed 209 proteins produced by A. amylolytica, which were mainly involved in carbohydrate transport and metabolism (43), amino acid transport and metabolism (23), protein turnover (18), cell wall/membrane/envelope biogenesis (16), energy production and conversion (10), and signal transduction (10), as shown in Supplementary Figure S2. Among these secreted proteins, 4 cellulases belonging to GH5 and GH6, 4 xylanases belonging to GH10, and two β-glucosidases belonging to GH1 were identified among the secretion proteins (Figure 3). These two β-glucosidases were designated AaBGL1 and AaBGL2.

According to the genome sequences of A. amylolytica YIM 77502^T, two putative genes encoding β-glucosidases AaBGL1A (GenBank: MH974516) and AaBGL2 (GenBank: MH974517) were amplified by PCR and introduced into the pET28a vector to construct recombinant plasmids pET28a-aabgl1 and pET28a-aabgl2. AaBGL1 and AaBGL2 consisted of 444 and 476 residues with theoretical molecular weights of 48.47 and 52.61 KDa, respectively. Signal peptides were not predicted at the N-termini of AaBGL1 and AaBGL2 based on analysis with Signal P 4.1 Server⁸. Sequence analysis revealed AaBGL1 and AaBGL2 contained distinct catalytic modules of glycosyl hydrolase family 1 (GH1) in the predicted enzyme proteins. The deduced amino acid sequence of AaBGL1 had the highest amino acid sequence identity (89%) to β-glucosidase (NCBI: WP 103938629.1) from Actinomadura echinospora. AaBGL2 had 84% similarity with β-glucosidases from Thermomonospora curvata (NCBI: WP 012852091.1) and Actinomadura echinospora (NCBI: WP 103937196.1). As shown in Figures 4A,B, the predicted three-dimensional model structures of AaBGL1 and AaBGL2 were very similar to other GH1 β-glucosidases with known structures The monomeric subunit of GH1 β-glucosidase adopts the expected topology of a single (α/β)₈ barrel, with additional units of quite well-conserved secondary structures inserted between the α/β units. A phylogenetic analysis of the protein sequences revealed AaBGL1 and AaBGL2 also clustered with β-glucosidases (Supplementary Figures S3, S4).

The β-glucosidase genes were successfully expressed in E. coli BL21 (DE3) and the resulting recombinant proteins with His-tagged N-termini were purified by Ni-NTA affinity chromatography. SDS-PAGE analysis indicated the molecular masses of the recombinant β-glucosidase proteins were in good agreement with the theoretical ones (Figures 4C,D).

To evaluate the potential use of AaBGL1 and AaBGL2 in degradation of lignocellulose, enzymatic hydrolysis of various lignocellulosic materials, including CMC, Avicel, and ionic liquid-pretreated birch sawdust and corncob powder, were performed. As shown in Figure 7, the glucose concentrations in the hydrolysis liquors were ordered Avicel > corncob > birch sawdust > CMC. After adding the β-glucosidases AaBGL1 and AaBGL2 to commercial cellulases, the degradation rates of CMC, Avicel, birch sawdust, and corncob powder increased by 37, 42, 33, and 9%, respectively. This suggests AaBGL1 and AaBGL2 cooperated with commercial cellulases in cellulose degradation.

An aerobic Gram-positive actinomycete, A. amylolytica YIM 77502^T can digest cellulose rapidly and efficiently. A large number of glycosidase enzymes, including 9 cellulase genes and 11 β-glucosidase genes, have been predicted in the genome of A. amylolytica. Other actinomycetes, such as Thermobifida fusca (Wilson, 2010), Thermobifida halotolerans YIM 90462^T (Yin et al., 2015), Thermoactinospora rubra YIM 77501^T (Yin et al., 2017), and Streptomyces strains (Ventorino et al., 2016), also display highly lignocellulose-degrading activities. To date, many cellulose-degrading enzymes have been identified from cellulolytic actinomycetes (Vanee et al., 2017), indicating cellulolytic actinomycetes, such as A. amylolytica, are good potential sources of cellulose hydrolases.

In this research, 4 cellulases and 2 β-glucosidases were identified from the secretome of A. amylolytica YIM 77502^T cultured with microcrystalline cellulose. These enzymes may be involved in the degradation of cellulose, but no signal peptides were found in the 2 identified β-glucosidases, AaBGL1 and AaBGL2. Broeker et al. (2018) also found 32 proteins lacking signal peptides in the secretome of Clostridium stercorarium. These 2 β-glucosidases of A. amylolytica found in the secretome may be secreted through the release of proteins from cells through cell lysis or alternative secretion pathways. The sequence similarity, tertiary structures, and ML phylogenetic trees of AaBGL1 and AaBGL2 suggest GH1 β-glucosidases are conserved. Discovery of AaBGL1 and AaBGL2 in the secretome of A. amylolytica indicates LC-MS/MS is potentially a highly useful method of discovering new proteins with specific functions.

During cellulose degradation, cellulolytic microorganisms secrete some hydrolytic enzymes, including free cellulases (endoglucanase, cellobiohydrolases, and β-glucosidases) and cellulosome, which consist of a wide variety of polysaccharide-degrading enzymes (e.g., cellulases, hemicellulases, and pectinases) (Wilson, 2009; Artzi et al., 2017). Most bacteria and fungi hydrolyze cellulose by secreting free cellulases (Cavedon et al., 1990; Ja’afaru, 2013). Meanwhile, a cellulosome is a supramolecular multienzyme complex that can efficiently degrade lignocellulose and is found in just a few bacterial species, including Clostridium (Ruminiclostridium) clariflavum and Clostridium (Ruminiclostridium) thermocellum (Bayer et al., 2004; Shiratori et al., 2009; Shinoda et al., 2018). As described above, A. amylolytica YIM 77502^T secreted cellulases extracellularly to hydrolyze cellulose. A total of 344 and 38 genes in the genome of A. amylolytica were found to potentially be involved in carbohydrate transport and secretory transport of proteins, respectively (Supplementary Figure S1). Of these genes, membrane transporters are membrane proteins associated with transportation of macromolecules, such as proteins, across biological membranes (Saier et al., 2006). ATP-binding cassette transporters (ABC transporters) are members of a transport system superfamily and involved in the translocation of various substrates, such as glucose, cellobiose, and galactose, across membranes (Kemner et al., 1997; Petit et al., 2015).

Membrane transporters and ABC transporters from A. amylolytica possibly transfer cellulose-degrading enzymes and carbohydrates. Through the activity of A. amylolytica extracellular enzymes, secretome and TLC plate analyses of CMC hydrolysis products, including endoglucanase, cellobiohydrolases, and β-glucosidases, revealed hydrolysis of cellulose to oligosaccharides (cellobiose as the main product) and monosaccharide (glucose). As shown in a schematic diagram (Figure 8), cellulose-degrading enzymes are secreted into the extracellular medium and the cellulosic substrate is degraded by synergistic hydrolysis of these free glycosidolytic enzymes. The cellulose hydrolyze these products (cellobiose and glucose), which are then transported to the cytoplasm across the cell membrane through ABC transporters. The oligosaccharides are eventually hydrolyzed to monosaccharides by intracellular β-glucosidases. Finally, glucose entrance into the Embden-Meyerhof-Parnas pathway and tricarboxylic acid cycle provided material (carbon source) and energy (ATP) required for the growth and reproduction of A. amylolytica. This is the process of cellulose hydrolysis in pure culture in the laboratory simulation, but cellulose does not exist in a pure state in nature. Besides cellulose, there are also large amounts of hemicellulose, lignin, and pectin in plant biomass. Xylan, one of the main components of hemicellulose, can be hydrolyzed by xylanases. As shown in Figures 3, 4 GH10 xylanases were identified in the secretome of A. amylolytica, indicating they were also induced by cellulose. Robison reported that Trichoderma reesei Rut C-30 produces extracellular xylanases when grown on cellulose (Robison, 1984). The complete hydrolysis of lignocellulose requires the synergistic cooperation of cellulases, xylanase, pectinase, laccase, and other enzymes (Van Dyk and Pletschke, 2012). Therefore, for A. amylolytica, the collaborative expression of cellulases and xylanases may be a functional adaptation for hydrolysing plant biomass to be used as a carbon source.

Two β-glucosidases (AaBGL1 and AaBGL2) secreted by A. amylolytica exhibited synergistic cooperation with commercial cellulases. After adding AaBGL1 and AaBGL2, the decomposition rate of Avicel (40%), CMC (37%), and ionic liquid-pretreated birch sawdust (33%) increased by more than 30%. Meanwhile, the degradation rate of ionic liquid-pretreated corncob improved only 9%. Compared to wood, such as birch sawdust, corncob contains more hemicellulose as it is more than one-third of the dry matter of the corncob (Sun et al., 2014). This indicates degradation of substrate by the synergistic action of different kinds of cellulases is related to the purity or amount of cellulose in the substrate. It was also suggested that cellulases need to cooperate with other enzymes, such as hemicellulases (e.g., xylanase) and pectinase, when lignocellulose is degraded. The importance of the cocktail method for degradation of lignocellulosic materials also indicates that more new enzyme resources need to be found to enhance this synergistic degradation (Ibrahim, 2011).

The enzymatic properties of AaBGL1 and AaBGL2 include that, like other β-glucosidases, their activities were completely inhibited by Cu²⁺ (Chen et al., 2010; Wu et al., 2018). However, the activity of AaBGL1 was not affected by 10 mM SDS. A strong denaturant of proteins, SDS can inactivate most enzymes (Joo and Chang, 2010). The SDS stability of AaBGL1 suggests it is suitable for application in industrial purposes. During cellulose hydrolysis by cellulases, the hydrolysis products inhibit the activity of the enzymes. Most β-glucosidases are sensitive to the final product of glucose, which limits the use of β-glucosidase and efficient degradation of cellulose. The inhibition constants (Ki) of AaBGL1 and AaBGL2 were 1502 and 193.5 mM glucose, respectively. Meanwhile, the Ki of most fungal β-glucosidases, such as β-glucosidases from Penicillium citrinum UFV1, Chaetomium globosum, and Neurospora crassa, are between 0.1 and 10 mM (Bohlin et al., 2010; Da Costa et al., 2016). This indicates AaBGL1 and AaBGL2 are glucose-tolerant β-glucosidases. The glucose-tolerances of β-glucosidases are of great significance due to increases in glucose concentration and the conversion rates of soluble fermentable sugars from cellulose degradation.