Ind. Eng. Chem. Res. 2006, 45, 4471-4474
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Microwave-Assisted Hydrothermal Degradation of Silk Protein to Amino Acids Armando T. Quitain,*,† Hiroyuki Daimon,‡ Koichi Fujie,‡ Shunsaku Katoh,† and Takashi Moriyoshi† Research Institute for SolVothermal Technology, 2217-43 Hayashi, Takamatsu, Kagawa 761-0301, Japan, and Department of Ecological Engineering, Toyohashi UniVersity of Technology, Tempaku, Toyohashi 441-8580, Japan
Microwave irradiation was applied to enhance the hydrothermal degradation rate of silk protein to amino acids. Results showed a significant increase in the yield of amino acids compared to the conventional heating method. Under microwave irradiation, the yield was favorable in NaOH compared to the commonly used HCl for protein hydrolysis. Moreover, this technique has also demonstrated its advantages in enhancing the recovery of tyrosine (Tyr), a known precursor of several neurotransmitters, from silk protein. obtained from digestion of the protein with chymotrypsin:
Introduction Utilization of silk protein as a natural source of biochemically important compounds such as amino acids has recently caught our interest. Silk protein consists of mostly glycine (Gly), alanine (Ala), serine (Ser), aspartic acid (Asp), and tyrosine (Tyr). These amino acids have wide uses and applications in pharmaceuticals, food products, animal nutrition, and cosmetic industries. These can be used to treat various diseases, such as renal, gastrointestinal, endocrinal, and dermal. In the food industry, amino acids are utilized as taste enhancers and animal feeds. These can also be reagents for the synthesis of new materials including electronic-related chemicals.1 The market forecast had shown that the global demand for amino acids would exceed $7.4 billion in 2004, and this demand is expected to further increase in the future.2 One possible method to obtain amino acids from proteins is by hydrothermal treatment. Our recent works on the application of hydrothermal treatment, utilizing its potential for hydrolysis reaction, have been mostly carried out on the recovery of amino acids from various proteinaceous wastes.3-6 However, results of these studies showed that pure water at elevated temperatures and pressures without any additives would not give a sufficient yield of amino acids for the process to be economically viable. On the other hand, it was reported that the application of microwave irradiation can enhance the rate of hydrolysis of proteins and peptides.7 This work investigates the use of microwave irradiation to enhance the hydrothermal degradation of silk protein, with emphasis on the production of amino acids, by elucidating the effects of various reaction conditions. Materials and Methods Silk Protein Sample. Dry silk protein powder was supplied by Gifu Bio-Industrial Institute (Gifu, Japan). Results of acid hydrolysis with 6N HCl solution showed silk protein to contain mostly Gly, Ala, Ser, Tyr, and Asp, at mole fractions of 27, 26, 19, 8, and 6% respectively. Lucas et al.8 have reported that silk protein consists mainly of the following repetitive sequence of amino acids found in the insoluble granular precipitate * Corresponding author. Tel.: +81-87-869-3440. Fax: +81-87-8693441. E-mail:
[email protected]. † Research Institute for Solvothermal Technology. ‡ Department of Ecological Engineering, Toyohashi University of Technology.
Gly-Ala-Gly-Ala-Gly-[Ser-Gly-(Ala-Gly)n]8Ser-Gly-Ala-Ala-Gly-Tyr (1) However, other amino acids such as Asp, Val, His, Pro, and Trp were also found in the soluble fraction. Method for the Study of the Initial-Stage Decomposition. Initial-stage decomposition of silk protein was first studied using a semibatch reactor equipped with a rapid sample-loading system. The details of the apparatus were reported elsewhere.9 In each run, ∼40 mg of sample placed inside a mesh capsule was loaded into the reactor. After loading the sample to the reactor, the first 96-cm3 effluent, equivalent to the volume of the pipe in the reactor downstream to the sampling port, was removed. The presence of organic compounds in the 96-cm3 effluent was then verified by measuring the total organic carbon (TOC) using Shimadzu TOC-5000A. Then, the sample was continuously collected for a period of 1 min, replacing the container after each sampling period. A detailed study was then carried out in a 6-mL batch reactor made up of SUS-316. The experiments were conducted over a temperature range of 200-300 °C, at corresponding saturated vapor pressures of 1.4-9 MPa and at reaction times up to 60 min. The sample-to-water ratio was fixed to ∼50 mg of sample in 5 g of deionized water. The reaction time started after reaching the desired temperature and pressure, usually after ∼2 min. After the desired reaction time had elapsed, the reactor was plunged into a water bath to cool it quickly to room temperature, thus ceasing any occurring reactions. Microwave-Assisted Reactor. Microwave-assisted reactions were carried out in a commercially available microwavedigestion system (model: Ultraclave, Milestone S. R. L., Italy), which uses a frequency of 2.45 GHz. In this apparatus, microwaves were generated by a magnetron system, and its irradiation power can be adjusted from 0 to 100% of a maximum 1000 W. The apparatus is linked to a personal computer, which controls the operating parameters (i.e., heating power, temperature, pressure, and reaction time). Experiments were conducted at a temperature range of 110200 °C and at various reaction times. In each experimental run, ∼50 mg sample and 10 mL of deionized water were placed in a 20-mL vessel made of poly(tetrafluoroethylene)/tetrafluorometoxil (PTFE/TFM). The vessel was sealed and placed in the microwave apparatus. The system was compressed up to a
10.1021/ie0580699 CCC: $33.50 © 2006 American Chemical Society Published on Web 05/18/2006
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Figure 1. Changes in amino acid composition in the effluents showing initial-stage decomposition behavior of silk protein at 250 °C (white box ) Asp, dot-shaded box ) Ser, white box with black dots ) Ala, diagonallined box ) Gly, and patterned box ) others).
pressure of 5 MPa by gradually injecting Argon gas. The microwave was then turned on to reach the desired reaction temperature. It took ∼20 min to reach a temperature of 200 °C, at a heating power of 1000 W. After the desired reaction time had elapsed, the reactor was cooled gradually to ∼50 °C before decompressing the reactor. To compare the results with those of the conventional heating method, the same experiments were carried out in a HU-25 Teflon reactor (San-Ai Kagaku, Japan) heated by a forced convection oven (FC-610, Advantec). Amino Acid Analysis. The amino acid contents of the reaction products were determined using a Shimadzu Amino Acid analyzer (Shimadzu Corp., Japan). The analyzer is equipped with an ion-exclusion column (ShimPack Amino-Na, Shimadzu Corp., Japan) and postcolumn labeling methods with a spectrophotometer (RF-10A, Shimadzu Corp., Japan). The sample was filtered with an ultrafiltration membrane (30 000 fractional molecular weight, Millipore Ultra Free C3) prior to analysis to maintain the good performance of the apparatus.
Figure 2. Behavior of Gly, Ala, Ser, and Asp formation from decomposition of silk protein under hydrothermal conditions (9 ) 200 °C, b ) 250 °C, and 2 ) 300 °C).
Figure 3. First-order plots of hydrothermal decomposition rate of Gly, Ala, Ser, and Asp.
Results and Discussion Behavior of Silk Protein Decomposition. The behavior of amino acid production from silk protein, during the initial stages of reaction at 250 °C, was investigated using the semibatch reactor. Results showed that Ser and Asp could be easily taken from the sequence of amino acids in proteins, as shown in Figure 1. The yield of Ser and Asp decreased, while the yield of relatively simple and low-molecular-weight amino acids such as Gly and Ala increased with time. The behavior of formation of Gly, Ala, Ser, and Asp was investigated in detail using the batch reactor. The dependence of formation of these amino acids on reaction temperature and time is summarized in Figure 2. Low temperature and short reaction time favored the formation of Ser and Asp. At 200 °C, while Ser increased, Asp decreased with time, reaching zero after 60 min. At 250 °C, Ser and Asp decreased, while Gly and Ala increased with time, similar to the trend at 200 °C. The increase in the amount of Gly and Ala at 250 °C is likely due to the high ion product of water, favoring the hydrolysis reaction. It is also likely that thermal degradation of Ser and Asp took place, thus lowering the yield. Comparison of molecular structures of these amino acids suggests a possibility of formation of Gly and Ala from Ser and Asp, with the increase in time and temperature. This has been investigated in our recent work on decomposition of amino acids in high-temperature and high-pressure water.10 At 300 °C, the yields of Gly and Ala decreased, apparently due to further decomposition to other organic compounds such as organic acids. Thus, experiments on the decomposition of
Figure 4. Comparison of the yield of four predominant amino acids from hydrothermal decomposition of silk protein at 200 °C, in 60 min between conventional and microwave-assisted heating methods.
amino acids were carried out at 300 °C. The first-order plots of decomposition of each amino acid, shown in Figure 3, illustrate that high-molecular-weight amino acids such as Ser and Asp decomposed faster than Gly and Ala. The results also suggest that the amount of amino acid in the products may serve as an indication of the extent of the degradation of silk protein, that is, a low Gly and Ala concentration would mean a low degradation rate and vice versa. This is taken as a basis in subsequent discussion related to the results of microwaveassisted degradation experiments. Microwave-Assisted Degradation. Microwave irradiation can be absorbed deep into the folding layers of silk protein to loosen the bonds, thus increasing the rate of hydrolysis. The use of microwave at 200 °C and a reaction time of 60 min, indeed, increased the yield by about 10-fold compared to the conventional heating method, as shown in Figure 4. The composition of amino acids shows that Ser and Asp are predominantly present in the products obtained using the conventional heating method, while Gly and Ala are mostly obtained using microwave. This result is in contrast with the
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Figure 5. Temperature-dependence of the yield of amino acids from silk protein at reaction time of 60 min in the presence of acid and alkali (diagonal-lined box ) Gly, white box with black dots ) Ala, dot-shaded box ) Ser, and white box ) Asp).
Figure 7. Effect of acid and alkali on the recovery of Tyr from silk protein by microwave-assisted reaction at 110 °C. Table 1. Comparison of the Yield of Tyr between Conventional and Microwave-Assisted (MW) Hydrothermal Degradation of Silk Protein yield (mg/(g of silk protein)) temp (°C)
time (min)
conventional
MW-assisted
150
30 60 120 30 60 120 30 60
0 0.05 0.15 0 0.13 0.76 0.08 0.49
0.18 0.37 0.56 1.03 1.34 2.42 2.26 3.70
175 200
Figure 6. Time-dependence of the yield of amino acids from silk fibroin at 110 °C in the presence of acid and alkali. (*The indicated reaction time is the time the reacting mixture is kept at a temperature of 110 °C. It takes ∼9 min for the reacting mixture to reach 110 °C at a microwave heating power of 800 W.)
results in Figure 1, which is an indication that the hydrolysis rate is slower in the conventional heating method compared to that in the microwave-assisted method. The same trends were observed at temperatures below 200 °C. These results illustrate the significant effect of microwave irradiation on the degradation rate of silk protein. Further experiments were carried out on the effect of acid (HCl) and base (NaOH) catalysts at temperatures of 100 and 200 °C, in a reaction time of 60 min. Results in Figure 5 show that addition of HCl and NaOH significantly increased the yield, even at a low temperature of 110 °C. The yield is favorable using NaOH compared with the commonly used HCl in proteindecomposition analysis. With NaOH, the result at 200 °C is almost identical with that at 110 °C, except for the absence of Ser, which likely underwent thermal decomposition. Thus, a relatively low temperature ∼110 °C is deemed suitable for protein decomposition to avoid thermal decomposition of some useful amino acids such as Ser. It may also be possible to shorten the reaction time even below 60 min. The microwave reactor apparatus was then programmed for the reaction mixture to ramp up from room temperature to 110 °C for 9 min at a maximum microwave heating power of 800 W. The indicated reaction time is the time that the reacting mixture is kept at a temperature of 110 °C. Experiments at 0 and 30 min were carried out, and the results are shown in Figure 6 in comparison with that in 60 min at two different concentrations of HCl and NaOH. Using NaOH, the yield is surprisingly high, even at a reaction time of “0 min”. The results confirmed the advantages of applying microwave irradiation to the degradation of silk protein, making the process more economical and efficient by operating at low temperature and short reaction time. More interestingly, experiments at
shorter microwave heating time at constant power should be carried out in our future studies. Enhanced Recovery of Tyrosine. One important amino acid present in silk protein is tyrosine (Tyr), a known precursor of several neurotransmitters such as dopamine. Some research findings showed its beneficial effect against Parkinson’s and Alzheimer’s diseases.11 The recovery of tyrosine (Tyr) from silk protein was also investigated by studying the effects of various parameters such as reaction temperatures and time and addition of acid and alkali. Table 1 shows the comparison of the yield of Tyr from silk protein by conventional and microwave-assisted hydrothermal treatments. The yield is defined as the amount of Tyr in the products per gram of silk protein. At 200 °C, in a reaction time of 1 h, the yield of 3.7 (mg of Tyr)/(g of silk protein) using microwave irradiation is significantly higher than that obtained by using the conventional heating method (0.49 (mg of tyrosine)/ (g of silk protein)). As shown in Figure 7, even at low temperature of 110 °C, the recovery significantly increased with the addition of HCl and NaOH. A maximum recovery of ∼81% was obtained in a reaction time of 30 min with 6 N NaOH. The decrease in Tyr recovery in 60 min was likely due to its thermal decomposition. A lower recovery of 53% was obtained at a higher temperature of 200 °C with 6 N NaOH in a reaction time of 60 min. Conclusion The application of microwave irradiation to enhance the hydrothermal degradation rate of silk protein to amino acids was investigated. At first, the initial stage hydrothermal decomposition of silk protein was elucidated to serve as a basis in the analysis of the results of microwave-assisted degradation. The effects of various reaction conditionsstemperature, time, and addition of acid and alkaliswere investigated. In general, the results showed the possibility of decomposing silk protein under hydrothermal conditions in the absence of any catalysts. Application of microwave irradiation enhanced
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the degradation rate. Addition of acid and alkali significantly increased the yield, and NaOH is more favorable for hydrolysis than the commonly used HCl in protein-decomposition analysis. The technique has also demonstrated its advantages in enhancing the recovery of tyrosine (Tyr), a known precursor of several neurotransmitters, from silk protein. This research lays the groundwork for further investigation on the microwave-assisted degradation of proteins under hydrothermal conditions, which can be useful for an economical and efficient recycling of proteinaceous wastes and materials for amino acid recovery. Acknowledgment This research was funded in part by the Japan Society for the Promotion of SciencesResearch for the Future Program Program (Causes and Effects of Environmental Loading and Its Reduction). Literature Cited (1) Ajinomoto’s Amino Acid Operations. http://www.ajinomoto.co.jp/ amino/e_aminoscience/index.html (last accessed April 2005). (2) World Amino Acids. http://www.the-infoshop.com/study/ fd5716_world_amino_acids.html (last accessed April 2005). (3) Kang, K.; Quitain, A. T.; Daimon, H.; Noda, R.; Goto, N.; Hu, H.; Fujie, K. Optimization of Amino Acids Production from Waste Fish Entrails
by Hydrolysis in Sub- and Supercritical Water. Can. J. Chem. Eng. 2001, 79, 65-70. (4) Daimon, H.; Kang, K.; Sato, N.; Fujie, K. Development of Marine Waste Recycling Technologies Using Sub- and Supercritical Water. J. Chem. Eng. Jpn. 2001, 34, 1091-1096. (5) Quitain, A. T.; Sato, N.; Daimon, H.; Fujie, K. Production of Valuable Materials by Hydrothermal Treatment of Shrimp Shells. Ind. Eng. Chem. Res. 2001, 40, 5885-5888. (6) Quitain, A. T.; Kang, K.; Sato, N.; Daimon, H.; Fujie, K. Behavior of Hydrothermal Decomposition of Silk Fibroin: A Model for Amino Acid Production from Proteinaceous Wastes. Presented at AIChE Annual Meeting 2001, Reno, Nevada, November 4-11, 2001. (7) Chen, S.-T.; Chiou, S.-H.; Chu, Y.-H.; Wang, K.-T. Rapid Hydrolysis of Proteins and Peptides by Means of Microwave Technology. Int. J. Pept. Protein Res. 1987, 30, 572-576. (8) Lucas, F.; Shaw, J. T. B.; Smith, S. G. Some Amino Acid Sequences in the Amorphous Fraction of the Fibroin of Bombyx mori. Biochem. J. 1962, 83, 164-171. (9) Kang, K.; Quitain, A. T.: Urano, S.; Daimon, H.; Fujie, K. Rapid Sample Injection in Semi-Batch Hydrothermal Treatment of Solid Wastes. Ind. Eng. Chem. Res. 2001, 40, 3717-3720. (10) Sato, N.; Quitain, A. T.; Kang, K.; Daimon, H.; Fujie, K. Reaction Kinetics of Amino Acid Decomposition in High-Temperature and HighPressure Water. Ind. Eng. Chem. Res. 2004, 43, 3217-3222. (11) Meyer, J. S.; Welch, K. M. A.; Deshmuckh, V. D. Neurotransmitter Precursor Amino Acids in the Treatment of Multi-Infarct Dementia and Alzheimer’s Disease. J. Am. Geriatr. Soc. 1977, 7, 289-298.
ReceiVed for reView August 10, 2005 Accepted March 30, 2006 IE0580699
