Zobellia sp. ZM-2 strain (CCTCC No. M2013256) is now preserved in the China Center for Type Culture Collection (Wuhan, China). P. pastoris GS-115 (Mut⁺) strain and plasmid pPIC9K were purchased from Invitrogen (Carlsbad, CA, United States). Luria-Bertani medium (LB) contained 5 g/L yeast extract, 10 g/L peptone, 10 g/L NaCl, and 100 μg/mL ampicillin (20 g/L agar was added for preparing solid medium). Minimal dextrose medium (MD) contained 13.4 g/L yeast nitrogen base (YNB) without amino acids (Solarbio, Beijing, China), 20 g/L glucose, 0.4 mg/L biotin, and 20 g/L agar. Yeast extract peptone dextrose medium (YPD) contained 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose (20 g/L agar was added for preparing solid medium). Buffered minimal glycerol medium (BMGY) contained 10 g/L yeast extract, 20 g/L peptone, 10 g/L glycerol, 3 g/L K₂HPO₄, and 11.8 g/L KH₂HPO₄.

The complete κ-carrageenase gene (cgkZ) sequence was obtained from the NCBI GenBank database (accession No. KC503903). Three pairs of primers were used to amplify the genes cgkZ, cgkZΔPst and cgkZΔCBM (Table 1). PCR was performed using Zobellia sp. ZM-2 genomic DNA as a template and high-fidelity Pfu DNA polymerase (Takara, Shiga, Japan). After the purification of the PCR products, the target genes and the vector pPIC9K were digested with AvrII and NotI (Thermo Fisher Scientific, Waltham, MA, United States). The digested products were purified by gel recycling, and the target gene and vector were ligated using T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, United States). The ligated product was transformed into DH5α competent cells. Selected positive colonies were cultured in LB medium containing 100 μg/mL ampicillin at 37°C for 12 h. The recombinant plasmids were extracted using a plasmid extraction kit (Omega, Winooski, VT, United States), and the plasmid sequences were determined by Sangon Biotech (Shanghai, China).

The recombinant plasmids pPIC9K-cgkZ, pPIC9K-cgkZΔPst and pPIC9K-cgkZΔCBM were linearized using SalI and transformed into P. pastoris by electroporation using a MicroPulser^TM electroporator (Bio-Rad, Hercules, CA, United States) at a voltage of 2 kV. After electroporation, the yeast cells were cultured in MD medium at 30°C for 3 days. Then the yeast cells were further diluted with sterile water and cultured on a YPD agar plate containing 4 mg/mL of geneticin (G418) at 30°C for another 3 days. The obtained colonies were activated in 10 mL of YPD medium at 28°C for 24 h, and then 2 mL of medium was transferred to a 1 L shaker flask containing 200 mL BMGY medium. P. pastoris was cultured at 28°C for 72 h with the addition of methanol (1%, v/v) every 24 h. After incubation, the culture was centrifuged and the supernatant was retained to measure κ-carrageenase activity. Enzymatic activity was measured as described by Liu et al. (2013) and protein concentration was measured using the Bradford method (Liu et al., 2011). Strains exhibiting high enzymatic activity were screened and stored at -80°C.

The genomic DNA of P. pastoris was extracted using a Yeast DNA Kit (Omega, Winooski, VT, United States). The copy number of each target gene was determined by real-time quantitative PCR using a 7900 HT real-time PCR system (Applied Biosystems, Foster City, CA, United States). A 10-fold series (10³–10⁸ copies) of linearized plasmids containing cgkZ and the endogenous gene (Glyceraldehyde 3-phosphate dehydrogenase, GAPDH), were used as templates to establish standard curves. The primers used in qPCR are listed in Table 1. PCR reactions were performed in a 20 μL mixture containing 10 μL of 2 × Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan), 0.3 μM of each primer, and 50 ng of DNA template. The PCR parameters were 95°C for 60 s, followed by 40 cycles at 95°C for 15 s, 58°C for 30 s, and 72°C for 60 s. Melting curve analysis was used to determine the specificity of amplification. For each target gene, the copy numbers was calculated based on the ratio of gene copies between target gene and endogenous control.

Fermentation conditions were optimized by modulating the temperature, pH, and methanol concentration. The optimum fermentation temperature was determined by testing several temperatures (22, 24, 26, and 28°C). The optimum pH was determined in media containing 20 mM of potassium phosphate at pH 5, 6, 7, or 8. Several concentrations of methanol (0, 0.5, 1, 1.5, and 2% v/v) were evaluated to determine the optimum concentration. The recombinant enzymes were obtained by fermentation under the optimal conditions for 120 h in a 1-L shaker flask containing 200 mL of BMGY medium. Methanol (1%, v/v) was added into the fermentation culture every 24 h. The enzymatic activity was measured every 12 h.

After incubation in shaker flask for 36 and 72 h, 1 mL of fermentation broth was used for the determination of transcription levels. Total RNA was extracted using a Yeast RNA Kit (Omega, Winooski, VT, United States). RNA was diluted with 50 μL of DEPC water and used for reverse transcription with ReverTra RT Master Mix (Toyobo, Osaka, Japan). The cDNA (25 ng) was used as a template to determine transcription levels by qPCR. Each qPCR reaction was performed as described above, with three biological replicates. The transcription level of each target gene was calculated based on the ratio of transcription levels between target mRNA and endogenous control (GAPDH).

The fermentation broth was centrifuged at 4000 × g for 20 min and the supernatant was concentrated by ultrafiltration using a 10 kDa membrane. Recombinant enzymes were purified by cation exchange chromatography using a CM-Sepharose Fast flow column (20 cm × 1.6 cm; GE Healthcare, Little Chalfont, United Kingdom), eluted first with 20 mM phosphate buffer (pH 6.5) for an elution volume of 30 mL, and then eluted with 20 mM phosphate buffer (pH 6.5) containing 1 M NaCl for an additional elution volume of 30 mL. Enzymatic activity and protein concentration were determined in samples obtained during purification. SDS-PAGE electrophoresis on a 12% (v/v) gel was performed to determine the molecular weights of recombinant enzymes after purification, according to the method described by Liu et al. (2011).

The optimum temperature for the purified enzymes was determined by measuring their activity at pH 6 and temperatures ranging from 30 to 65°C. Thermal stability was determined as the residual activity after pre-incubating the enzymes for 0–4 h at several temperatures (40, 42.5, 45, 47.5, or 50°C). The activity of the enzymes pre-incubated at 4°C was regarded as 100%. The optimum pH for each purified enzyme was determined by measuring the enzymatic activity at 50°C in 20 mM phosphate buffer with a pH ranging from 4 to 10. To determine the effects of ions and chemical reagents, enzymatic activity was measured in solutions containing 5 mM of Cu²⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, Fe³⁺, EDTA, SDS, dithiothreitol (DTT); 10, 50, and 100 mM of Na⁺, K⁺; 0.5% (v/v) of TritonX-100, Tween-80, methanol, and glycerol, respectively. Enzymatic activity was measured at 50°C and pH 6.0. The kinetic parameters of the purified enzymes were determined in solutions containing κ-carrageenan at concentrations between 1 and 10 mg/mL at 50°C, pH 6.0 for 5 min. The Michaelis constant (K_m) and V_max were determined from Lineweaver–Burk double reciprocal plots using Origin Pro 8.0 (OriginLab, Northampton, MA, United States).

Oligosaccharides were prepared by the enzymatic hydrolysis of 10 mg/mL κ-carrageenan solution. Hydrolysis was performed in the presence of 1 mL of recombinant enzyme at 40°C for 6 h. The final product was precipitated by adding five volumes of 95% ethanol. Negative-ion electrospray ionization mass spectrometry (ESI-MS) was performed on a Q-TOF mass spectrometer (Waters, Milford, MA, United States) to analyze the oligosaccharides. The sample was dissolved in acetonitrile/water (1:1, v/v) and 5 μL of the solution containing the dissolved sample was injected for analysis with 1 mM ammonium bicarbonate /acetonitrile (1:1, v/v) as the mobile phase at a flow rate of 10 μL/min.

The results of fermentation optimization and enzymatic characterization experiments were analyzed by the standard deviation method, using SPSS 18.0 (IBM, New York, NY, United States). All original data represented three biological replicates. The data presented in tables are expressed as means ± standard deviations.

The gene encoding the full-length κ-carrageenase, cgkZ is 1638-bp in length, whereas the genes encoding the truncated cgkZΔPst and cgkZΔCBM are 1200 and 948-bp in length, respectively (Figure 1A). The results of agarose gel electrophoresis suggested that the target genes were well amplified from the genomic DNA of Zobellia sp. ZM-2 (Figure 1B). Based on the functional annotations of the cgkZ gene in the NCBI Genbank database, the Pst was truncated in cgkZΔPst and the CBM was truncated in cgkZΔCBM, whereas their catalytic domains were retained. The expression vector pPIC9K used in this study contains α-factor signal sequence that can promote the secretion of recombinant proteins. The recombinant vectors pPIC9K-cgkZ, pPIC9K-cgkZΔPst and pPIC9K-cgkZΔCBM were transformed into P. pastoris after linearization. The gene encoding histidinol dehydrogenase (HIS4) provides a selectable marker to isolate recombinant strains from MD plates, and the kanamycin resistance gene allows the selection of high activity colonies from YPD agar plates containing 4 mg/mL of G418 (Figure 1C). These strains were preserved for an analysis of gene copy number and the results are shown in Table 2. The qPCR results showed a linear relationship between the gene copy number and C_t value. The slope and R² of the amplification standard curve for cgkZ were -3.082 and 0.995, respectively, while those for the endogenous control gene were -2.794 and 0.991, respectively. Based on the standard curve, the copy numbers of gene cgkZ, cgkZΔPst, and cgkZΔCBM were approximately 7, 7 and 6, respectively.

In this study, the enzymatic activities of the recombinant extracellular enzymes were evaluated at four different temperatures (Figure 2A). The three recombinant enzymes exhibited high levels of activity below 24°C. When the fermentation temperature reached 28°C, the relative enzymatic activities decreased to 52–60%, indicating that high fermentation temperatures inhibit the secretion of recombinant enzymes. The effect of pH on recombinant enzymatic activity is shown in Figure 2B. The results show that the optimum fermentation pH for enzymatic activity was 6–7, which was favorable for the production of the recombinant enzymes. When the fermentation pH was 5, the relative enzymatic activity decreased to approximately 73%. The effect of methanol concentration on enzymatic activity is shown in Figure 2C. At methanol concentrations of 0.5–1% (v/v), large amounts of the recombinant enzymes were secreted extracellularly, and they showed high activity levels. At the methanol concentration of 1.5% (v/v), the relative enzymatic activity decreased to 82–86%.

The curve for recombinant enzyme activity is shown in Figure 2D. Recombinant strains were cultured in a 1-L shaker flask at 22°C, pH 6, and 1% (v/v) methanol. From 0 to 24 h, methanol was not added and no enzymatic activity was detected. With the first addition of methanol (24 h), methanol was used as the sole carbon source by the recombinant strains and κ-carrageenase secretion was observed. The maximum activity of the recombinant enzymes was detected at 96 h of incubation. The maximum activities for the recombinant enzymes cgkZ, cgkZΔPst, and cgkZΔCBM were 4.68, 5.70, and 3.02 U/mL, respectively.

The results for gene transcription levels as measured by qPCR are shown in Table 3. The transcription levels of cgkZ, cgkZΔPst, and cgkZΔCBM were significantly higher at 36 h than at 72 h. Compared with the transcription level of cgkZ, the transcription levels of cgkZΔPst and cgkZΔCBM increased to 2.9- and 3.5-fold at 36 h, and to 4.3- and 2.8-fold at 72 h, respectively. Since the gene copy numbers of cgkZ and cgkZΔPst were similar, the higher enzymatic activity of cgkZΔPst was due to its higher transcription level. This result indicated that truncation of the Pst increased the expression of the recombinant enzyme. However, the enzymatic activity of cgkZΔCBM was depressed even though cgkZΔCBM showed a higher transcription level than cgkZ, indicating that the truncation of the CBM may decrease the enzymatic activity of κ-carrageenase.

The supernatants obtained from cultures grown under shaker-flask fermentation were concentrated before cation exchange chromatography. As shown in Table 4, after cation exchange chromatography, the specific activity of cgkZ increased by 9.7-fold to 356.74 U/mg, while the specific activities of cgkZΔPst and cgkZΔCBM were 487.78 and 265.66 U/mg, representing 10.4- and 11.0-fold increases, respectively. The SDS-PAGE result of the purified recombinant κ-carrageenase is shown in Figure 3. The molecular weights of cgkZ, cgkZΔPst and cgkZΔCBM were found to be approximately 65, 45, and 40 kDa, respectively. The molecular weights of these proteins significantly differed and most of the impurities were removed after cation exchange chromatography.

In this study, the temperature and pH ranges selected for optimization experiments were chosen based on the enzymatic characterization of κ-carrageenase produced by Zobellia sp. ZM-2, which had an optimum temperature and pH of 39°C and 6.0, respectively (Liu et al., 2013). The effect of temperature on recombinant κ-carrageenase activity expressed in P. pastoris is shown in Figure 4A. The recombinant enzyme cgkZ showed the highest activity at 50°C and its relative activity decreased to 89% at 55°C. The recombinant enzymes cgkZΔPst and cgkZΔCBM showed the highest activities at 55°C and their relative activities decreased to 84 and 88% at 60°C, respectively.

In the experiment for the determination of the enzymes’ thermal stabilities, after incubation at 45°C for 4 h, approximately 50% of the enzymatic activity was retained (Figure 4B). When the recombinant enzymes were incubated at 42.5°C for 4 h, more than 90% of the enzymatic activity was retained (data not shown). All the recombinant enzymes were stable below 42.5°C. Approximately 50% of the enzymatic activity was lost after 1 h of incubation at 47.5°C and after 30 min of incubation at 50°C (data not shown). These results demonstrate that these recombinant enzymes expressed by P. pastoris have a higher thermal stability than one that was previously expressed by E. coli, which was stable below 35°C (Liu et al., 2013). κ-Carrageenase obtained from Pseudoalteromonas porphyrae showed thermal stability at 30°C (Liu et al., 2011), which was also lower than the thermal stabilities of the recombinant enzymes expressed in this study.

The effect of pH on recombinant κ-carrageenase activity is shown in Figure 4C. The recombinant enzyme cgkZ showed the highest activity at pH 7 and its optimum pH was 6–7. The recombinant enzymes cgkZΔPst and cgkZΔCBM showed the highest activities at pH 6.0, with an optimum pH of 5–7. For recombinant enzyme cgkZ, at pH 4 or 8, the relative activity decreased to approximately 76%, indicating that it began to become inactivated under those conditions.

The effects of ions and chemical reagents on recombinant κ-carrageenase activity are shown in Table 5. The results show that Na⁺ promoted the activity of recombinant κ-carrageenase, while a high concentration of K⁺ inhibited enzymatic activity. Mg²⁺ and Ca²⁺ stimulated the enzymatic activity slightly, while Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ strongly inhibited the activity. Among the tested chemical reagents, the addition of DTT stimulated enzymatic activity to 178–217%, which was similar to the reports of Yao et al. (2012) and Liu et al. (2013). SDS significantly inhibited the enzymes. EDTA had slight inhibitory effects on the recombinant enzymes. TritonX-100, Tween-80, methanol, and glycerol had minimal effects on the activities of the three recombinant enzymes.

Based on the Lineweaver–Burk double reciprocal plots, the K_m values of cgkZ, cgkZΔPst and cgkZΔCBM were 2.07, 1.85, and 1.04 mg/mL, respectively, which were higher than that reported by Liu et al. (2013) (0.84 mg/mL). Compared with the κ-carrageenase obtained from P. porphyrae (K_m 4.4 mg/mL), these recombinant enzymes showed higher substrate affinities (Liu et al., 2011). The V_max values of these recombinant enzymes were 0.77, 1.35, and 0.50 mg/mL⋅min, respectively.

Although the recombinant enzymes were encoded by target genes with different lengths, the end-products after the enzymatic hydrolysis of κ-carrageenan were nearly the same. The MS spectrum is shown in Figure 5. Two intense fragment peaks were observed. An ion at m/z 394 (z = 2) with a molecular weight of 790 Da represented a tetrasaccharide with two [A-G_4S] repeating units ([A-G_4S]₂). Another ion at m/z 391 (z = 3) with a molecular weight of 1176 Da represented a hexasaccharide with three [A-G_4S] repeating units ([A-G_4S]₃) (Yu et al., 2017). Some other weak peaks were also observed. The results of MS showed that the final products of enzymes cgkZ, cgkZΔPst and cgkZΔCBM-catalyzed hydrolysis were mainly composed of κ-carrageenan tetrasaccharide and hexasaccharide, which is similar to the results obtained by Liu et al. (2013). Although the Pst and CBM were truncated in cgkZΔPst and cgkZΔCBM, respectively, the catalytic properties of these enzymes did not change compared with those of the full-length recombinant enzyme.