Construction of the metagenomic library
Macroalgae samples (Grateloupia filicina, Chondrus ocellatus, and Scagassum) were collected from Halmahera Island (0o 36'N, 127o 52'E), Indonesia. The samples were washed with sterilized seawater, then cut into pieces and placed in a sterile tube. Sterile water was added and shaken three times with a vortex oscillator for 2 min each time. Then, sterile filter membranes (50 mm, 0.22-μm pore size) were used to collect the bacteria for later use. A FastDNA Spin Kit for Soil (MP Bio) was used to extract the genomic DNA from the collected bacteria samples. After testing, the qualified genomic DNA was sent to Jingneng Biotechnology Co., Ltd. (Shanghai, China) for sequencing, assembly, and functional annotation.
Plasmids, vectors, and substrates
Plasmid pET-30a (+) and E. coli BL21 (DE3) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Soluble starch, agarose, carrageen, sodium cellulose, and alginate were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, china). Grateloupia filicina, Chondrus ocellatus, and Scagassum were collected from Yangkou Beach in Qingdao, China.
Gene synthesis and sequence analysis of Amy2587
The metagenomic data of the macroalgae-associated bacteria samples were analyzed and the amy2587 gene sequence was obtained by screening the metagenomic library and used as the template to synthesize the target gene. The gene was synthetized by Nanjing Kingsley Biotechnology Co., Ltd. (Nanjing, China). BLAST Search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to identify the amy2587 sequence and the DNAMAN software package (http://www.lynnon.com/) was used for multiple sequence alignment. And the motifs were analyzed using Motif Search (http://www.genome.jp/tools/motif/).
Expression of amy2587 and purification of Amy2587
The amy2587 gene and pET-30(a) were digested by BamHI and XbaI endonucleases respectively, then ligated by T4 DNA ligase to construct the recombinant plasmid Amy2587+pET-30(a). The recombinant Amy2587 was expressed in E. coli BL21 (DE3). The obtained transformants were incubated on LB medium (50 μg/ml kanamycin) with constant shaking at 150 rpm at 37°C. Isopropyl-β-D-thiogalactopyranoside (IPTG) (0.5 mM) was added to express the fusion protein when the OD600 reached 0.6. After induction for 16 h at 16°C, the cells were collected, placed on ice, and crushed using an ultrasonic cell crushing apparatus.
An Ni-NTA His Tag Kit (Novagen) was used to purify the recombinant Amy2587. First, binding buffer (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) was used to wash the recombinant Amy2587, then elution buffer with different concentrations of imidazole (20 mM, 80 mM, 140 mM, and 200 mM) was used to elute the recombinant Amy2587 (Riera et al. 2003). Finally, the target protein Amy2587 was assessed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Blakesley and Boezi 1977).
Substrate specificity of Amy2587
To determine the multifunctionality of Amy2587, we studied its substrate specificity. 100 μl purified Amy2587 (0.2 mg/ml) and 900 μl substrate (0.1% soluble starch, 0.1% agarose, 0.1% carrageen, 0.1% sodium cellulose, and 0.1% alginate) were incubated for 40 min at 50°C, and the Amy2587 activity was measured by the 3,5-dinitrosalicylic acid (DNS) method (Chi et al. 2014).
Characterization of Amy2587
To determine the effect of pH on Amy2587 activity, 100 μl purified Amy2587 (0.2 mg/ml) and 900 μl substrate (0.1% soluble starch, 0.1% agarose, 0.1% carrageen, 0.1% sodium cellulose, and 0.1% alginate) were incubated in different buffer systems from pH 4.0–11.0 (pH 4.0−7.0, Na2HPO4-citric acid; pH 7.1−8.9, Tris-HCl; pH 9.0−10.6, glycine-NaOH) for 40 min at 50°C. Amy2587 activity was determined by the DNS method. The highest detected enzyme activity was defined as 100%.
To determine the effect of temperature on Amy2587 activity, 100 μl purified Amy2587 (0.2 mg/ml) and 900 μl substrate (0.1% soluble starch, 0.1% agarose, 0.1% carrageen, 0.1% sodium cellulose, and 0.1% alginate) were incubated at 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 40 min. The highest detected enzyme activity was defined as 100%.
To determine the thermostability of Amy2587, 100 μl purified Amy2587 (0.2 mg/ml), which had been pre-incubated at 40°C, 50°C, and 60°C for 0 to 24 h, and 900 μl substrate (0.1% soluble starch, 0.1% agarose, 0.1% carrageen, 0.1% sodium cellulose, and 0.1% alginate) were incubated for 40 min. The highest detected enzyme activity was defined as 100%.
To determine the effect of metal ions (2 mM) on Amy2587, metal cations (Sr2+, Ni2+, Ca2+, Ba2+, Mn2+, Mg2+, Fe2+, Fe3+, K+, Cu2+, Na+) were added to the corresponding reaction mixtures and incubated for 40 min at 50°C and pH 7.0, the standard assay conditions. The enzyme activity in the absence of metal ions was defined as 100%.
Kinetic parameters assay
The concentrations of the five substrates in the assay system were changed, and changes in the enzymatic reaction rates were measured under the standard assay conditions. The Lineweaver-Burk double-reciprocal method (Morrison 2002) was used to obtain the kinetic parameters and determine the kinetic behaviors of Amy2587.
Variant assay of Amy2587
To explore the multifunctionality of Amy2587, a motif search program was used to analyze the sequence. Three truncates of Amy2587 were constructed in which the α-amylase_N, α-amylase, and Glyco_hydro_66 motifs were each deleted separately; the truncates were named amy2587a (1428 bp), amy2587b (918 bp), and amy2587c (1491 bp), respectively. The three genes were cloned and heterologously expressed in E. coli BL21 cells.
Raw seaweeds digestion using Amy2587
We employed one-step enzymatic digestion method to obtain oligosaccharides directly from raw seaweed using the novel multifunctional α-amylase Amy2587 as follows. Purified enzyme solution (0.2 mg/ml) was added to artificial seawater containing 2% (w/v) of dried red seaweeds, Grateloupia filicina, which mainly produces carrageenan, and Chondrus ocellatus, which mainly produces agar, and dried brown seaweed, Scagassum, which mainly produces alginate. The reaction mixtures were incubated for 0.25 h, 0.5 h, 1 h, 2 h, 6 h, 12 h, and 24 h at 50°C under the constant shaking at 150 rpm. The ability of Amy2587 to digest the three raw seaweed substrates was demonstrated by measuring the reducing sugar content using the 3,5-dinitrosalicylic acid (DNS) method (Kang and Kim 2015).
GenBank accession number
The complete open reading frame of Amy2587 has been deposited in the GenBank database under accession number MW839461.