Extraction and Mw determination of fucoidan
The fucoidan (FPs) was extracted by the acidic method in 0.1 and 0.2 M HCl to achieve the maximum yield of fucoidan. Figure 3A showed that different ranges of temperatures (50, 70, and 90 °C) and time (1, 3, and 5 h), applied with 0.1 and 0.2 M HCl. The extracted fucoidan was reacted with 2 M TFA to further hydrolyze the fucoidan. According to the plot as mentioned earlier, an optimum yield (6.5% DW) was attained by using 0.1 M HCl at 90 °C after 3 h, while 4.0% of yield was obtained on 0.2 M HCl at the same processing conditions. The results also elaborated that the yield was decreased as the time interval increase at the same temperature, so the duration of hydrolysis also have a significant impact on yield (Fig. 3A). Previous reports mentioned that about 7.1% DW of fucoidan was extracted by using acid hydrolysis at 90 °C and after 3 h [23] while, about 3.4, 1.8, and 2.8% of fucoidan were reported from U. pinnatifida by using different extraction methods [24–26].
The Mw of HF was determined by GPC, and the results revealed that the polysaccharide had an Mw of 1,109 kDa (Table 1). The Mw obtained was higher than the fucoidan extracted previously by chemical (1,035.5 kDa) [27] and hot water (262 kDa) [28] treatments.
Compositional and structural analysis of fucoidan
The composition of monosaccharides present in fucoidan was analyzed by HPAEC-PAD, and the results demonstrated that there were various proportion of different monosaccharides (Table 1) such as galactose (21.8%), mannose (6.7%), fucose (35.9%), arabinose (1.5%), glucuronic acid (26.2%), and rhamnose (0.05%). The fucoidan extracted from the same species have different compositions; the difference might be due to growing conditions, environmental changes, region, analytical process, and extraction methods [27–29]. The literature exposed that fucose was the main monosaccharide present in fucoidan, however other monosaccharides were also reported, but the composition of fucose vary among the same species (Table 1). This variation was due to the extraction methods that have a significant influence on yield and compositional properties [24–26, 28, 30].
The structural changes of hydrolyzed and non-hydrolysed polysaccharides can be observed by FTIR spectrum (Fig. 3B), it is an imperative technique to describe and classify the functional or chemical groups existing in different samples [31]. A characteristic band of non-hydrolysed fucoidan (NHF) was shown at 3,419.3 cm− 1 which corresponds to O-H deformation [11], while for hydrolyzed fucoidan (HF) the O-H band shifted at higher wavenumber (3453.8 cm− 1). The small peaks at 2,927.3 and 2,925.3 cm− 1 were assigned as stretching vibration of C-H bonds of NHF and HF, respectively [11, 32]. The fractions appeared in HF at 1,667.4–1,704.2 cm− 1 may indicate the occurrence of acylamino groups [33]. A small vibrational peak at 2,927.3 cm− 1 was assigned to O-H frequency for NHF although this peak was shifted to low number at 2,925.3 cm− 1 for HF [32, 34]. The occurrence of CH2 and CH3 was indicated by finding the peaks at 1,442.8 and 1,236.3 cm− 1 (Fig. 3B) [32]. The peaks (HF) observed at 3,418 and 2,875 confirmed the existence of OH and CH group of fucose at the C-6 position [34].
Table 1. Comparison of molecular weight and composition of monosaccharides in fucoidan extracted from U. pinnatifida by different methods
Extraction method
|
Yield (%)
|
Monosaccharide composition
|
Mw (kDa)
|
References
|
Fucose (%)
|
Galactose (%)
|
Glucose (%)
|
Mannose (%)
|
Xylose (%)
|
Arabinose (%)
|
Glucuronic acid (%)
|
Rhamnose (%)
|
Acid hydrolysis
|
6.5
|
35.9
|
21.8
|
4.1
|
6.7
|
3.7
|
1.5
|
26.2
|
0.05
|
1109.7
|
Present study
|
HCl extraction
|
3.4
|
53.0
|
38.0
|
2.0
|
5.0
|
2.0
|
NR
|
NR
|
ND
|
NR
|
[24]
|
Hot water extraction
|
NR
|
NR
|
NR
|
23.81
|
4.26
|
64.0
|
5.90
|
NR
|
NR
|
1035.52
|
[27]
|
Anion exchange chromatography
|
0.5 – 1.8
|
59.0
51.0
|
30.0
48.0
|
1.0
|
8.0
1.0
|
2.0
|
NR
|
NR
|
ND
|
NR
|
[25]
|
Chemical extraction
|
16.6 – 43.4
|
52.0 – 72.5
|
27.5 – 39.0
|
NR
|
3.6 – 8.9
|
NR
|
NR
|
NR
|
NR
|
262
|
[28]
|
Ethanol extraction
|
NR
|
78.8
|
21.
|
NR
|
NR
|
NR
|
NR
|
NR
|
NR
|
23600 - 5200
|
[30]
|
CaCl2 extraction
|
2.8
|
16.4
|
NR
|
NR
|
NR
|
NR
|
NR
|
NR
|
NR
|
171
|
[26]
|
NR: not reported; ND: not displayed
Amino acid sequence analysis of recombinant Pa-LFI
The whole-genome from P. rhizosphaerae was examined and assigned the GenBank accession No. WP_076167696.1 with 596 number of amino acids having isoelectric point 5.06 and characterized as L-fucose isomerase (L-FI). The hypothetical amino acids sequence of recombinant Pa-LFI exhibited 76% identity with A. pallidus (accession No. WP_063389244.1), 73% with both T. toyohensis (accession No. WP_084665866.1) and C. polysaccharolyticus (accession No. WP_035172025.1), while 69% and 64% with D. turgidum DSM6724 (accession No. YP_002352328.1) and E. coli W (accession No. ADT76410.1), respectively.
To investigate the amino acids sequence of recombinant Pa-LFI, the sequence alignment tool ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) was applied to relate the recombinant Pa-LFI from earlier reported bacteria. The secondary structure of A. pallidus (WP_063389244.1) with PDB ID: 3A9T was determined by the ESPript server (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) [35] and verified by (http://services.mbi.ucla.edu/SAVES/) server.
The comparison between the sequence alignment of different previously characterized L-fucose isomerases (L-FIs) was mentioned in Figure S1. The multiple sequence alignment of P. rhizosphaerae (WP_076167696.1) was compared with A. pallidus (WP_063389244.1), C. polysaccharolyticus (WP_035172025.1), D. turgidum DSM6724 (YP_002352328.1), T. toyohensis (WP_084665866.1) and E. coli W (ADT76410.1). All of these sequences displayed approximately 65% similarity among themselves. The secondary structure premeditated from the previously discovered 3D structure of A. pallidus (WP_063389244.1) with PDB ID: 3A9T using ESPript server [35]. The figure S1 represents strictly conserved residues with a red background, the highly conserved residues epitomize with red type in blue boxes, while the symbol stars above the residues show secondary structure of recombinant Pa-LFI which attained from L-FI from A. pallidus (PDB ID: 3A9T).
Expression and purification of the recombinant Pa-LFI
The truncated Pa-LFI sequence was commercially synthesized with restriction endonuclease sites Ndel and Xhol and an in-frame 6 × histidine tag sequence, which cloned into the pET-22b(+) an expression vector. The recombinant plasmid was emulated and expressed into E. coli BL21 (DE3) cells. The non-characterized recombinant Pa-LFI protein was purified by single-step nickel affinity chromatography through electrophoretic homogeneity. The purified protein of recombinant Pa-LFI exhibited 33-folds of purification with 104.5 U mg− 1 of specific activity and 86% yield for L-fucose. The recombinant Pa-LFI presumed expression yield in E. coli cell culture 4,695 U L− 1 for L-Fucose, which is higher than 2,360 U L− 1 from T. toyohensis [16]. The specific activity of recombinant Pa-LFI was sophisticated than that of 85.5, 93 and 76 U mg− 1 from T. toyohensis, D. turgidum and C. saccharolyticus, respectively [16, 18, 19].
The recombinant purified Pa-LFI protein formed a single thick band of protein around 65 kDa on SDS-PAGE, called subunit molecular mass, which is equal to theoretical value 6,5439.90 Da analyzed by using a server of ExPASy-Compute pI/Mw tools (Fig. 4A). The molecular mass was consistent with the predicted values of recombinant Pa-LFI, which revealed that the recombinant Pa-LFI was successfully overexpressed. The native molecular mass was deliberated by using HPLC with a single peak at a retention time of 6.87 min in elution profile possessed a molecular mass of around 396 kDa under nondenaturing conditions by comparing with various reference standards (Fig. 4B). The theoretical molecular mass of one unit of recombinant Pa-LFI is around 65 kDa, and the native molecular mass was estimated to be 396 kDa, corresponding to six subunits. The results elaborate that recombinant Pa-LFI is a homohexamer, parallel to the previously characterized L-fucose isomerase from D. turgidum [19].
Effect of pH, temperature, and metal ions on the activity of recombinant Pa-LFI
The influences of pH on the activity of recombinant Pa-LFI was examined at 50 °C, and different pH ranges from 5.0 to 10.0, using three buffers system: Na-phosphate (pH 5.0–7.0), Tris-HCl (pH 7.0–9.0) and glycine-NaOH (pH 9.0–10.). The recombinant Pa-LFI exhibited optimum activity on pH 6.5 in Na-phosphate buffer (Fig. 5A). The activity increased in Na-phosphate buffer pH 6.5, and it decreased rapidly as the pH increased from 6.5 to 10.0 in Tris-HCl and glycine-NaOH buffers. While in Tris-HCl buffer, the pH behavior for the enzyme was first raised to 7.5 and then suddenly decreased to 9.0. Glycine-NaOH showed the lowest activity in decreasing pattern, presenting that recombinant Pa-LFI was more sensitive to higher pH than lower pH. Lower pH reactions are favorable to industrial application because they inhibit the browning reactions and diminish unwanted by-products [36]. The enzyme activity maintained higher level under weak acidic conditions at pH 6.5, which is consistent with other L-FIs such as C. saccharolyticus (7.0) [18], D. turgidum (7.0) [19] and lower than T. toyohensis (9.0) [16].
The enzyme stability was assessed by incubating purified recombinant Pa-LFI at 4 °C for 24 h (Fig. 5B), and 50 °C for 1 h (Fig. 5C) with different pH ranges from 5.0–10.0 in above three buffers. The recombinant Pa-LFI fully sustained its activity from pH 5.0–10.0 which revealed no significant effect on its activity at 4 °C for 24 h, while incubating at 50 °C for 1 h the activity increased from the optimum which shows the thermal effect of pH on enzyme stability.
The influences of temperature on the activity of recombinant Pa-LFI was evaluated in Na-phosphate buffer at pH 6.5, temperature ranging from 35–70 °C (Fig. 5D). The optimum temperature for recombinant Pa-LFI was determined as 50 °C. More than 85% of relative activity was maintained between 40–55 °C. This result indicates that 50 °C is a favorable temperature for the isomerization of L-fucose which is higher than E. coli B/r (37 °C) [37], lower than C. saccharolyticus (75 °C) [18], T. toyohensis (75 °C ) [16] and D. turgidum (80 °C) [19]. Generally, higher temperatures are required for industrial applications, to produce functional sweeteners because high temperature can reduce the risk of microbial contaminations, enhance the speed of reaction, convert the reaction equilibrium towards products and increase solubility [38].
The influences of metal ions on recombinant Pa-LFI activity were studied after removal of previously existing metal ions from the purified protein with the help of EDTA to achieve precise results. Afterward, the EDTA was removed with Tris-HCl buffer (pH 7.0) by subsequently dialyzing the enzyme. The recombinant Pa-LFI is metalloenzyme in which metals ions entertain as a cofactor for the isomerization process of rare sugars. The enzyme was incubated with ten different divalent metals ions (EDTA, Ni2+, Fe2+, Zn2+, Mn2+, Ba2+, Ca2+, Co2+, Mg2+, and Cu2+) at a final concentration of 1 mM to examine their effects on the enzyme activity at pH 6.5 and 50 °C (Fig. 5E). The optimum metal ion for recombinant Pa-LFI was Mn2+ (450%) while Co2+ (261%) also enhanced the activity after Mn2+. Numerous divalent metal ions, including Ni2+, Zn2+, Ba2+, Ca2+, and Mg2+ enhanced more than 100% activity of recombinant Pa-LFI. In contrast, EDTA, Fe2+, and Cu2+ exert a negative effect on the catalytic activity of the recombinant enzyme.
Thermostability and melting temperature ( T m ) of recombinant Pa-LFI
The thermostability of recombinant Pa-LFI was measured at different ranges of temperatures from 30, 40, 50, 60 and 70 °C for 0, 4, 8, 12, 16, and 20 h by determining the residual activity (Fig. 5F). The recombinant enzyme revealed good thermostability at a temperature under 40 °C and retained more than 60% of residual activity than that of 50 °C when incubated for 20 h. Nevertheless, the residual activity of recombinant Pa-LFI was significantly decreased when the incubated temperature increases from 40 to 70 °C. However, the residual activity decreased to 4% and 7% at 60 and 70 °C after 12 h and 8 h respectively (Fig. 5F). The recombinant Pa-LFI was more than 50% active after 12 h on its optimum temperature (50 °C), which is the useful property of this enzyme. According to the first-order kinetics, the half-lives (t1/2) at different temperatures were measured to be 70 h (30 °C), 25.4 h (40 °C), 12.6 h (50 °C), 5.3 h (60 °C) and 2.1 h (70 °C), which is quite higher than previously reported C. saccharolyticus having 62 h, 13 h, 6 h, 2 h and 1 h at 60, 65, 70, 75 and 85 °C [18] and for D. turgidum 20 h, 12 h, 7 h, 5 h and 2 h at 65, 70, 75, 80 and 85 °C [19].
DSC delivers another significant property for the structural stability of recombinant Pa-LFI, which provides a differential heat flow as a transformation of temperature. The melting temperature (Tm) is a distinguished property to analyze the structural stability of the protein. The Tm of recombinant Pa-LFI examined by nano-DSC was measured to be 75 °C (Figure S2), which is higher than the optimum temperature (50 °C) and valuable for industrial application. The Tm of L-FI from C. polysaccharolyticus was 80.3 °C having 55 °C of optimum temperature, which was sustained with the present findings [17].
Specificity of substrates and kinetic parameters of recombinant Pa-LFI
The specific activities of recombinant Pa-LFI were inquired for L-fucose, D-arabinose, D-altrose, L-galactose, and L-xylose (Table 2). The recombinant Pa-LFI isomerization reaction displayed only a single product against each substrate and L-fucose was found to the optimum substrate with 104.5 ± 1.15 U mg-1 of the specific activity. The specific activities for D-arabinose, D-altrose, and L-galactose were exhibited to be 91.9 ± 1.77, 26.8 ± 0.76 and 2.3 ± 0.29 U mg-1, respectively followed by L-fucose (Table 2). There were lowest activities detected against L-galactose, and no activity was for L-xylose because hydroxyl groups existing in aldose substrates on the left-handed configuration at C2 and right-handed C3 and C4 positions such as L-fucose, D-arabinose, D-altrose, and L-galactose. All these results specify that L-fucose was the optimum substrate for recombinant Pa-LFI. These results further verified that recombinant Pa-LFI belongs to L-FI family similar to D. turgidum (93 U mg-1) [19], higher than T. toyohensis (85.5 U mg-1) [16], C. saccharolyticus (76 U mg-1) [18], E. coli B/r (64 U mg-1) and E. coli K-2 (63 U mg-1) [37].
Table 2
Substrate specificity and Kinetic parameters of recombinant Pa-LFI
Substrate | Product | Specific Activity (U mg− 1) | Km (mM) | Kcat (min− 1) | Kcat/Km (mM− 1 min− 1) |
L-Fucose | L-Fuculose | 104.5 ± 1.15 | 86.2 ± 2.3 | 32831 ± 20 | 335 ± 3.5 |
D-Arabinose | D-Ribulose | 91.9 ± 1.77 | 67.5 ± 2.1 | 19782 ± 43 | 293 ± 1.7 |
D-Altrose | D-Allulose | 26.8 ± 0.76 | 57.3 ± 1.8 | 2275 ± 9 | 40 ± 2.8 |
L-Galactose | L-Tagatose | 2.3 ± 0.29 | ND | ND | ND |
L-Xylose | L-Xylulose | 0.0 ± 0.00 | ND | ND | ND |
ND: not displayed |
The kinetic parameters of recombinant Pa-LFI for L-fucose, D-arabinose, and D-altrose were investigated by the nonlinear-regression method under optimum conditions of reaction (pH 6.5 and 50 °C) (Table 2). L-fucose was the optimum substrate for recombinant Pa-LFI, and it showed Km 86.2 ± 2.3 mM, which is higher than D-arabinose, D-altrose because of the isomerization rate higher for L-fucose. The Kcat/Km values were 335 ± 3.5, 293 ± 1.7 and 40 ± 2.8 (mM-1 min-1) for L-fucose, D-arabinose and D-altrose, respectively (Table 2). The results elaborate that L-fucose from recombinant Pa-LFI showed lower affinity but higher activity than that of D. turgidum [19]. A comparison of enzymatic characteristics and kinetic parameters of recombinant Pa-LFI with other microorganisms are given in Table 3.
Table 3
Comparison of enzymatic properties and kinetic parameters of recombinant Pa-LFI
Microorganisms | Molecular weight (kDa) | Temperature (˚C) | Optimum pH | Specific Activity (U mg− 1) | Metal ions | Km (mM) | Vmax (U mL− 1) | Reference |
P. rhizosphaerae | 66 | 50 | 6.5 | 104.5 | Mn2+ | 86.2 | 9.62 | Present work |
T. toyohensis | 66 | 75 | 9.0 | 85.5 | Mn2+ | 81.2 | 3.7 | [16] |
C. polysaccharolyticus | 65 | 55 | 6.5 | 108.2 | Mn2+ | 94.2 | 9.6 | [17] |
D. turgidum | 68 | 80 | 7.0 | 93 | Mn2+ | 90 | 2.3 | [19] |
E. coli B/r | 65 | 37 | 8.0 | 64 | Co2+ | 42 | 0.25 | [37] |
E. coli K-2 | 65 | 37 | 8.0 | 63 | Co2+ | 45 | 0.26 | [37] |
C. saccharolyticus | 68 | 75 | 7.0 | 76 | Mn2+ | 141 | 0.25 | [18] |
Production of L-fuculose commercial fucose and fucoidan by recombinant Pa-LFI
L-fuculose was produced from commercial L-fucose (Sigma, St. Louis, MO, USA) and hydrolyzed FPs by recombinant Pa-LFI in a reaction mixture of 0.5 mL at pH 6.5 and 50 °C. Upon the addition of 30, 50 and 100 g L-1 of commercial L-fucose (Sigma-Aldrich) (Fig. 6A) and HF (Fig. 6B), the enzymatic reaction attained its equilibrium state at 4, 5 and 6 h, demonstrating conversion rates of 30.32, 30.27, and 30.29% and 5.80, 5.80, and 5.66% respectively. During the high level of L-fuculose production by recombinant Pa-LFI, 9.0, 15.13 and 30.27 g L-1 and 1.74, 2.90 and 5.7 g L-1 L-fuculose was obtained from 30, 50 and 100 g L-1 of L-fucose and HF respectively. As compare to the previously described bacteria, C. polysaccharolyticus (28%) pH 6.5 at 55 °C [17], T. toyohensis (24.9%) pH 9.0 at 75 °C [16], C. saccharolyticus (24%) pH 7.0 at 70 °C [19] and K. pneumonia (10%) pH 9.0 at 40 °C [39] produced L-fuculose. Although, we used a different concentration of L-fucose and HF to produce L-fuculose still the conversion rates were similar and closed to 30% and 5.66% respectively.
After a comparison of all other L-fuculose producing bacteria, it exhibited that recombinant Pa-LFI produced a higher conversion ratio (30%) of L-fuculose from L-fucose with no byproducts and 5.8% from HF, which was a natural and cheap source from seaweed of U. pinnatifida. It was well distinguished and convenient for the industrial processes of recombinant Pa-LFI for the production of highly valuable L-fuculose. It could simplify the process of purification method and reduced the cost of downstream industry.