Sonicator

Cell free extract was prepared as described by Bellou et al. [48 (link)]. Briefly, yeast cells were washed twice with 50 mM Na₂HPO₄/KH₂PO₄ buffer (pH 7.5), and re-suspended at a ratio of 1 mL buffer per 0.5 g wet biomass, which were disrupted on ice using sonicator (Scientz, Ningbo, China), and the debris was removed through centrifugation. The supernatant was collected and filtered through 0.2 μm membrane, and the activities of IDH (EC 1.1.1.42), ACL (EC 4.1.3.8), MAE (EC 1.1.1.40) and ICL (EC 4.1.3.1) were determined, respectively, according to methods developed previously [49 (link)–52 (link)]. The protein content was determined by the Coomassie blue staining method [53 (link)]. The analytical results were an average of triplicate measurements.

Primer pairs in Table S4 were used for the amplification of the putative agarase genes aga3420 using a C1000 Thermal Cycler (Bio-Rad, California, CA, USA). The PCR program was as follows: 3 min at 95 °C; 25 cycles of 95 °C for 15 s; 55 °C for 15 s; 72 °C for 1 min/kb; and an extension at 72 °C for 6 min. The PCR products were further purified using a Universal DNA Purification Kit (Tiangen Biotech, Beijing, China). The purified PCR products were ligated with the pEAZY^®-Blunt E2 expression vector (TransGen, Beijing, China). The recombinant vector was transformed into the competent cells of E. coli BL21(DE3) pLysS.
The E. coli BL21(DE3) pLysS cells harboring the recombinant vector were cultured at 37 °C in LB broth (5.0 g yeast extracts, 10.0 g peptone, 10.0 g NaCl, and 1 L deionized water) with additional 100 μg/mL ampicillin. The culture broth was induced by 0.1 mM IPTG at 16 °C for 18 h until the OD600 value reached 0.6–0.8. The cells were collected by centrifugation at 10,000 rpm for 10 min at 4 °C, and the cells were fractured using a sonicator (Scientz, Ningbo, China). The Ni-nitrilotriacetic acid (NTA) Sefinose™ Resin (Tiangen Biotech, China) was used to further purify rAga3420 with 6 × His-tagged from the crude enzyme solution. The molecular weight and purity of rAgaW1540 were subsequently estimated using SDS-PAGE and Coomassie brilliant blue staining.

The E. coli BL21(DE3) pLysS cells harboring the recombinant vector were cultured at 37 °C in LB broth (5.0 g yeast extracts, 10.0 g peptone, 10.0 g NaCl, and 1 L deionized water) including 100 μg/mL ampicillin, and the culture broth was induced by adding 0.1 mM IPTG at 16 °C until the OD_600nm reached 0.6–0.8. The cells were collected by centrifugation at 10,000 rpm for 10 min at 4 °C, and then were subsequently resuspended in a Tris-HCl buffer (10 mM; pH 7.4). The resuspended cells were fractured using a sonicator (Scientz, Ningbo, China) at a rate of 3s/2s impulse frequency for 20 min in ice. The Ni-nitrilotriacetic acid (NTA) Sefinose™ Resin (Tiangen Biotech, Beijing, China) was used to further purify rAgaW1540 with 6×His-tagged from the crude enzyme solution. The molecular weight and purity of rAgaW1540 were subsequently estimated by SDS-PAGE and Coomassie brilliant blue staining.