Effects of Different Extraction and Purification Methods on the Acquisition of Phycoerythrin from Porphyridium purpureum

Yuanchao XU Shengxin NIE Zhen LIU Jinglong LI

Abstract Phycoerythrin， as the main lightharvesting antenna in Porphyridium purpureum， exists at the outermost end of the phycobilisome. It has advantages of good fluorescence intensity， antioxidation， scavenging free radicals， and high chroma， so it has been widely used in food， cosmetics， pharmaceuticals， and other industries. In this study， the effects of different extraction （ultrasonic breaking method， bead grinding method， liquid nitrogen grinding method， and freezingthawing method） and purification methods （salting out method， ultrafiltration method， and combination of salting out and ultrafiltration method） on the acquisition of phycoerythrin from P. purpureum were studied， and the characteristics of phycoerythrin in the P. purpureum were identified. The results showed that the freezingthawing method could extract phycoerythrin from the powder of P. purpureum to the utmost extent， and the concentration of the extracted phycoerythrin was up to 0.036 g/L. The salting out method could most effectively purify phycoerythrin， and the purity index was 2.216. The identification of phycoerythrin by ultraviolet absorption spectroscopy and fluorescence spectroscopy indicated that the phycoerythrin had the maximum absorption peak at 545 nm， and the maximum Stokes shift was up to 79 nm. Due to its high fluorescence characteristics， it can be used as a fluorescent marker in the fields of molecular biology and clinical medicine， and can also be used as a good photosensitizer in tumor therapy.

Key words Phycoerythrin; Extraction; Purification; Identification

Porphyridium purpureum （also called Porphyridium cruentum） is a primitive only singlecelled red alga that was first discovered by Naegeli in 1849. P. purpureum has the characteristics of high biomass and yield and rich high valueadded products in many microalgae strains. P. purpureum cells can synthesize a variety of bioactive substances during growth， such as phycobiliproteins （phycobiliproteins are divided into phycoerythrin PE， phycocyanin PC， allophycocyanin APC）， and polysaccharides from P. purpureum （mainly referring to extracellular sulfate lipopolysaccharide）， polyunsaturated fatty acids （mainly referring to arachidonic acid ARA and eicosapentaenoic acid EPA）. Among them， phycoerythrin， a high valueadded product produced from P. purpureum， has been widely used in food， cosmetics， pharmaceuticals and other industries. Because of its high fluorescence， high light stability， high fluorescence quantum yield and large Stokes shift[1-2]， it can be used as a fluorescent marker in molecular biology， clinical medicine and other fields[3]. Because it is safe and natural， it also has great potential as a good photosensitizer. For example， as a photosensitizer， it can be applied in photodynamic therapy of tumors. However， due to the limitation of production level and production technology， the yield of phycoerythrin is not high， and the extraction and purification are difficult， which limits the largescale production of phycoerythrin. In this study， effects of different extraction and purification methods on the acquisition of phycoerythrin from P. purpureum were studied， and the production program of phycoerythrin was optimized to suit largescale production.

Materials and Methods

Culture of P. purpureum

The P. purpureum used in this experiment was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology （FACHB） and cultured in artificial seawater medium （ASW）[4].

Collection of P. purpureum

Firstly， 3 ml of P. purpureum liquid that was cultured for 18 d was put in a centrifuge tube and centrifuged for 10 min at a speed of 1 200 g. After the supernatant was discarded， the algae were washed twice with deionized water. The precipitate was collected by centrifugation. The precipitate was divided into two parts， of which a part was freezedried on a freeze dryer for 12 h and then ground into algal powder for phycoerythrin extraction， and the other was used as alga slurry for phycoerythrin extraction.

Different extraction methods of phycoerythrin

Freezingthawing method

At first， phycoerythrin was extracted by the modified freezingthawing method[5]. That is， 3 ml of deionized water was added to centrifuge tubes containing the alga slurry and alga powder respectively， and the centrifuge tubes were frozen in a refrigerator at -20 for 2 h， and then thawed at room temperature for 1 h. After they were   repeatedly frozen and thawed three times， the supernatant was collected after centrifugation.

Liquid nitrogen grinding method

Phycoerythrin was extracted by the modified liquid nitrogen grinding method[6]. The prepared alga slurry and alga powder were respectively ground in liquid nitrogen for 10 min， during which liquid nitrogen was continuously loaded， and then 3 ml of deionized water was added to the samples. Finally， the supernatant was collected after centrifugation.

Ultrasonic breaking method

To extract phycoerythrin by the ultrasonic breaking method， 3 ml of deionized water was added to centrifuge tubes containing the alga slurry and alga powder respectively， and then an ultrasonic breaker probe was inserted into the samples on ice to break them for 5 s. After they were repeatedly broken three times， the supernatant was collected after centrifugation.

Bead grinding method

To extract phycoerythrin by the modified beadmilling method， 1.5 ml of deionized water was added to centrifuge tubes containing the alga slurry and alga powder respectively， and then small magnetic beads （ 0.05 and 0.10 mm accounted for 50% respectively） were added to the centrifuge tubes according to the volume ratio of 1/5 （v/v）. The centrifuge tubes were shaken at high speed for 15 s on a highspeed vibrating bead mill， and then placed on ice for 30 s. After breaking was repeated three times， 1.5 ml of deionized water was added to them. The supernatant was collected after centrifugation.

Different purification methods of phycoerythrin

Pretreatment of phycoerythrin crude extract

Crude phycoerythrin was extracted by the above freezingthawing method. The phycoerythrin solution obtained by freezing and thawing was Filtered through a 0.22 m membrane and stored as a control group at 4.

Ultrafiltration methodThe phycoerythrin crude extract was first purified by ultrafiltration method[9]. The phycoerythrin crude extract was added to a 50 kDa ultrafiltration centrifuge tube， and then the centrifuge tube was centrifuged for 15 min at a speed of 4 500 g. After the phycoerythrin solution in the centrifuge tube was taken out， 1 ml of deionized water was added to the centrifuge tube to collect residual phycoerythrin together. The residual phycoerythrin was washrf twice， and the final volume was 3 ml. It was stored at 4.

Salting out method

The salting out method[10]is a classic method of protein purification. To purify phycoerythrin by the optimized salting out method， ammonium sulfate was added to the phycoerythrin crude extract to make its saturation reach 65%. They were stirred evenly and placed in water bath at 25 for 6 h. After it was centrifuged for 10 min at a speed of 1 200 g， the precipitate was collected， and 3 ml of deionized water was added to redissolve it. It was preserved at 4.

Combination of ultrafiltration and salting out method

The phycoerythrin crude extract was purified by the above salting out method firstly and then by the above ultrafiltration method. The obtained phycoerythrin solution was preserved at 4.

Identification of phycoerythrin

Determination of phycoerythrin by ultraviolet spectrophotometer

The ultraviolet absorption spectrum （200-900 nm） of phycoerythrin was measured with a UV2600 ultraviolet spectrophotometer produced by Japanese Shimadzu， and the ultraviolet absorption values of phycoerythrin at 455， 565 and 592 nm were measured with a UV1750 ultraviolet spectrophotometer produced by Japanese Shimadzu. Phycoerythrin concentration was detected by using the formula of Beer and Eshel[11]as follows：

PE=[（OD565 nm-OD592 nm）-（OD455 nm-OD592 nm）≠0.2]≠0.12

where PE is phycoerythrin concentration （g/L）; OD is the optical density of phycoerythrin solution at a specific wavelength.

Determination of phycoerythrin by fluorescence spectrophotometer

The photoluminescence （PL） spectrum of phycoerythrin solution was measured with an F7000 fluorescence spectrophotometer produced by Japanese Hitachi with a 150 W xenon lamp as an excitation source. The formula of Stokes shift is as follows：

Results and Analysis

Comparison of different extraction methods

Seen from Fig. 1， in group D1， the alga powder was difficult to maintain steady state in liquid nitrogen， and collided sharply after liquid nitrogen was added to the alga powder， so the alga powder was not suitable for phycoerythrin extraction by liquid nitrogen grinding.

As shown in Fig. 1， the concentrations of phycoerythrin extracted by different extraction methods were different. The concentration of phycoerythrin extracted from the alga powder or alga slurry was the highest in group C1 or C2， showing that the freezingthawing method could extract phycoerythrin from P. purpureum as much as possible. According to Table 1 and Table 2， the concentration of phycoerythrin was 0.036 g/L in group C1 and 0.031 g/L in group C2. It was the lowest in groups B1and B2， only 0.009 and 0.008 g/L respectively， so the bead grinding method was not ideal when it was used to extract phycoerythrin. In addition， the state of P. purpureum had certain effects on the concentration of phycoerythrin extracted by the same method. Seen from the comparison of groups C1 and C2， the alga powder was more conducive to the extraction than the alga slurry. The difference between groups C1 and C2 was 0.005 g/L.

Therefore， the best method for phycoerythrin extraction from P. purpureum was the freezingthawing method， and the best state of P. purpureum was powder. Under the optimal conditions， the maximum concentration of phycoerythrin could reach 0.036 g/L. On the contrary， the bead grinding method was not ideal when it was used to extract phycoerythrin from P. purpureum， and the liquid nitrogen grinding method caused huge loss to the powder of P. purpureum.

Comparison of different purification methods

Ultraviolet absorption spectra of phycoerythrin solution purified by different methods are shown in Fig. 2. The maximum ultraviolet absorption value of phycoerythrin solution was at 545 nm， and A565 nm is the characteristic absorption peak of phycoerythrin solution， while A620 nm and A650 nm were the characteristic absorption peaks of phycocyanin and allophycocyanin respectively[12]， indicating that the P. purpureum contains not only a large amount of phycoerythrin （PE） but also a certain amount of phycocyanin （PC） and allophycocyanin （APC）[13]. The purity is usually determined as the ratio of A565 nm to A280 nm， which defines the relationship between the presence of phycoerythrin and other contaminating proteins[14]. Some researchers used the ratio A615/A565 to determine the purity of phycoerythrin and phycocyanin， as phycocyanin is the closest contaminating protein to phycoerythrin[15].

As shown in Fig. 2， The absorbance of phycoerythrin in the crude extract was the highest at 565 and 280 nm. According to Table 3， the concentration of phycoerythrin in the crude extract was 0.135 mg/mL， but the purity was only 1.103. It indicates that there were more miscellaneous proteins in the crude extract， and the concentration decreased， while the purity improved significantly after the purification by ultrafiltration. The salting out method could directly double the purity of phycoerythrin in the crude extract to 2.216， while the concentration dropped to 0.111 mg/ml， and the recovery rate was 82%. The saltingout method had a better purification effect on the phycoerythrin solution than the ultrafiltration method. The combination of ultrafiltration and salting out method did not greatly improve the purity of the phycoerythrin solution， but the recovery rate was lower; the loss became larger， and the concentration was only 0.088 mg/ml. Therefore， the salting out method is considered to be a simple and effective purification method.

Characteristics and characterization of phycoerythrin

The ultraviolet absorption spectrum and photoluminescence spectrum of the phycoerythrin solution were determined according to the above method， and its Stokes shift was calculated according to the spectroscopy of phycoerythrin.

The ultraviolet absorption spectrum of the purified phycoerythrin solution is shown in Fig. 3， and the representative ultraviolet absorption wavelength at 400-700 nm was selected. Two characteristic absorption peaks （A565 nm and A545 nm） and characteristic absorption shoulder （A499 nm） of phycoerythrin were labeled. There was a small absorption peak at 620 nm， which referred to the characteristic absorption peak of phycocyanin. Phycocyanin is often used as a contaminating protein of phycoerythrin. The effect of phycocyanin on the purity of phycoerythrin should be removed as much as possible. The fluorescence emission spectrum of the phycoerythrin solution at an excitation wavelength of 499 nm is shown in Fig. 4. The fluorescence emission wavelength of 500-700 nm was selected， and the maximum emission peak was at 578 nm. Studies have shown that whether any absorption wavelength of phycoerythrin （499， 545 and 565 nm） is used as an excitation wavelength， the fluorescence emission peak of phycoerythrin is at 780 nm， which also indicates the fluorescence stability of phycoerythrin. According to the Stokes shift formula： ┐┡=┡em-┡ex=578 nm-499 nm=79 nm， it is calculated that the maximum Stokes shift of the purified phycoerythrin from P. purpureum could reach 79 nm.

Rhodamine B， as a synthetic dye， has been usually used as a food additive[16]， but it has been experimentally proven that rhodamine B is carcinogenic， so it is now not allowed for food dyeing. Phycoerythrin， a natural harmless dye can replace traditional food additives for food dyeing.

Conclusions and discussion

Seen from the effects of different extraction methods on the extraction of phycoerythrin from the alga slurry and alga powder， the freezingthawing method could extract the phycoerythrin from the alga powder to the utmost extent. The phycoerythrin solution extracted by the freezingthawing method was used as a crude extract to compare different purification methods. The results showed that the salting out method could effectively purify the phycoerythrin solution with high purity. The ultraviolet absorption spectrum， concentration， fluorescence emission spectrum， Stokes shift and fluorescence quantum yield of the purified phycoerythrin solution were measured， and the results showed that the phycoerythrin had a highly stable fluorescence quantum yield and Stokes shift up to 79 nm and is natural and nontoxic， so its research value is very huge.

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