Controlled conversion of proteins into high molecular weight peptides

techniques are highly-specialized and require tedious synthesis, isolation and. 44 ... amino acids in HTW at long reaction times (t = 0-60 min). ... t...
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Research Article pubs.acs.org/journal/ascecg

Controlled Conversion of Proteins into High-Molecular-Weight Peptides without Additives with High-Temperature Water and Fast Heating Rates Taku Michael Aida,*,† Minori Oshima,† and Richard Lee Smith, Jr.†,‡ †

Graduate School of Environmental Studies and ‡Research Center of Supercritical Fluid Technology, Tohoku University, Sendai, Aramaki Aza Aoba 6-6-11, Aoba-ku, Sendai 980-8579, Japan S Supporting Information *

ABSTRACT: Reaction of bovine serum albumin (BSA) protein in hightemperature (200−260 °C) water (HTW) with fast heating rates (ca. 135−180 K·s−1) without acid or base additives gives high-molecular-weight (1500−8300 Da) peptides with minimal formation of amino acids and ammonia. The decrease in the number-average molecular weight of peptides after HTW treatment of BSA could be described by a kinetic model based on random scission mechanism of the polymer chain. Reaction of BSA in HTW under identical conditions with slow heating rates (ca. 0.25 K·s−1) gives peptides of low molecular weight with formation of amino acids and ammonia for which the kinetics could not be described by a random scission mechanism. The activation energy determined for the conversion of BSA into high-molecular-weight peptides with fast heating rates in high-temperature water was 16.4 kJ·mol−1. Reaction of proteins in high-temperature water with fast heating rates inhibits initial aggregation that occurs during slow heating rates and allows controlled conversion of the denatured polymer chain into high-molecular-weight peptides. KEYWORDS: Amino acids, Biomass, Biopolymers, Hydrothermal, Peptides, Reaction kinetics, Reaction mechanism, Subcritical water

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INTRODUCTION Peptides are molecules that consist of 10−50 amino acid monomers that are linked by amide bonds and are derived from the modification of proteins. Peptides have wide application in the medical field for delivering molecules into target cells,1,2 in materials science fields for designing self-assembled building blocks for mechanical and electronic devices3,4 and in food and health fields for improving food-safety and increasing food product shelf life.5,6 Although peptides can be produced from proteins with molecular biology, the techniques are highly specialized and require tedious synthesis, isolation, and purification steps often requiring large amounts of organic solvents that also generate much solid waste.7 Large-scale production of peptides on a 1000 kg-scale per year will require development of new approaches.8,9 In wastewater treatment systems, much of the organic nitrogen that enters into the environment is due to proteins that are incompletely removed by the treatment process or are generated as a result of biological treatment.10 Previously, we showed that organic nitrogen available in wastewater treatment effluents could be recycled as nutrients for microalgae cultivation with hydrothermal treatment and proposed that energy of the process could be obtained from low-quality (150 to 200) °C waste heat.11 Further, we found that spent microalgae could be recycled for microalgae cultivation with hydrothermal treatment and that high amounts of water-soluble proteins would become available.12 Thus, the wastewater treatment process of many different chemical industries could © 2017 American Chemical Society

provide the raw materials required for biorefineries that produce useful high molecular weight peptides from proteincontaining waste streams. High-temperature water (HTW) refers to a liquid state condition of water that is at temperatures above 200 °C but generally below its supercritical condition.13 The dissociation of water in its liquid state increases with increasing temperature from ambient conditions, so that acid-catalyzed reactions such as hydrolysis are promoted in high-temperature water without the addition of acid catalysts.14 Research on HTW treatment of proteins15 has mostly focuses on obtaining amino acids,16−20 while there are few reports on the production of peptides.21−25 Rogalinski et al.17 studied the decomposition of BSA protein in HTW at short reaction times (t = 4−180 s) using a flow reactor for the purpose of forming amino acids and reported a kinetic model. Sunphorka et al.18 studied the decomposition of rice bran protein into amino acids in HTW at long reaction times (t = 0−60 min). In that work, the authors observed the aggregation of protein at low temperatures (80 °C) and proposed a twostep hydrothermal kinetic method where the protein first aggregates then out of solution and then finally decomposes into amino acids. The aggregation of proteins is a physical change of the protein molecule that is triggered by changes in the solvent Received: April 13, 2017 Revised: July 26, 2017 Published: July 28, 2017 7709

DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715

Research Article

ACS Sustainable Chemistry & Engineering

Water was fed into the apparatus at a flow rate of 10−20 mL·min−1 by an HPLC pump. The stream was then preheated by an electric furnace and ribbon heater before it was introduced into the reactor from the mixing cross tee. The BSA solution (1 wt %), which was stored at room temperature, was fed into the reactor at the mixing cross tee from a different line (Figure S1). The ratio between the mass flow rates of the preheated water and BSA solution was 2:1. The concentration of BSA solution after mixing at the reactor was 0.33 wt %. The reaction temperature was achieved by mixing the BSA solution and preheated water at the mixing cross tee. The reaction proceeded as the solution flowed through the reactor and was terminated as it was rapidly cooled by the double-pipe heat exchanger. The solution was depressurized at the back pressure regulator and the product effluent was filtrated with a membrane filter (1 μm) to obtain a solid and a liquid filtrate. The liquid filtrate was defined as liquid product. The solid was further dried and weighed by the same method given previously and defined as solid product. The reaction temperature and pressure investigated in flow experiment was 200 to 260 °C and 25 MPa. The mean residence time of the experiments varied from 0.3 to 9.3 s depending on the reactor length (reactor volume) without changing the total flow rate. The mean residence time (τ) of the solution was calculated by the following equation:

environment such as pH, temperature, and pressure.26 Aggregation of proteins starts from the unfolding of the natural protein, that is followed by the intramolecular refolding of the unfolded protein forming new physical cross-links within and between protein molecules.27,28 The hypothesis examined in this work is that the physical structure of the protein molecule during the hydrolysis is key to understanding its controlled decomposition. Although the production of peptides from proteins using hydrothermal methods has been studied for many reaction conditions, most reports use batch-type reactors, where the reaction time is on the order of minutes.29,30 However, it is probable that the reaction kinetics of decomposition is on the order of seconds rather than minutes and during the heating period, protein aggregation must occur concurrently. Therefore, treatment of protein with HTW at fast-heating rates and precise short reaction times could allow one to distinguish between these kinetic processes. Further, if the two processes (protein decomposition and protein aggregation) can be distinguished, then a kinetic model can be developed that allows prediction of the molecular weight of the product peptides. The objective of this work was to investigate the conversion of BSA protein in high-temperature water using fast heating rates for the purpose of producing high molecular weight peptides. Reaction kinetics are evaluated and a kinetic model that provides the molecular weight of the product is developed.

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τ(s) = V ×

ρT,P Ftotal

(1)

where V (cm3) is the reactor volume, ρT,P (g·cm−3) is the water density at temperature T and pressure P, and Ftotal (g·s−1) is the sum of the two flow rates, solution and water, at each experiment condition. The deviation of the flow rates of water and BSA solution at the reactor was kept to within 5% of each flow rate, which corresponds to about 5% error in the mean residence time. Temperature measurement of the fluid was taken at a point 5.8 cm after the mixing cross tee (Figure S1). For all experiments, the reaction temperature was achieved at this point. The time required for the solution to reach reaction temperature was below the mean residence time at this point and was calculated to be below 1.3 s, for all reaction conditions. Therefore, the heating rate for the flow experiments was 135−180 K·s−1. The data given are averages and deviations of samples obtained from three individual experiments. Analyses. The molecular weight distribution of proteins in the liquid product was analyzed using a SEC system and the numberaverage molecular weight and weight-average molecular weight values were evaluated. The SEC system consisted of a packed column (PROTEIN KW-G, KW-802.5, SHODEX), HPLC pump (PU-2080, JASCO), auto sampler (AS-2055 Plus, JASCO), column oven (CO2065 Plus, JASCO), and UV detector (UV-970, JASCO). The temperature in the column oven was 45 °C, and the flow rate of the pump was 1.0 mL·min−1. The wavelength of the UV detector was set to be 280 nm. The mobile phase was a phosphate buffer solution prepared by equal molar of sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate 12-water, and sodium chloride buffer having a phosphate concentration of 0.1 M and sodium chloride concentration of 0.15 M. A calibration curve was constructed using protein standards of known molecular weights (Table S1). The total nitrogen concentration in the liquid product was measured by TOC/ TN analyzer (TOC-VCHS TNM-1, Shimadzu Corp.). The concentration of proteins in the liquid product was quantified by a BCA method.32 The amino acid concentration in the liquid product was measured by ninhydrin method.33 The ammonia concentration in the liquid product of the batch and flow experiments was quantified with an ammonium ion meter (TiN9001, TOKO Chem. Works.). The protein, amino acid, ammonia, residue yields, and nitrogen recovery were evaluated on a nitrogen basis of the initial BSA solution. Theory. The reaction kinetics of BSA in high-temperature water were evaluated by fitting a kinetic model34 to the obtained experimental data. The kinetic model34 assumes that the decomposition of a linear polymer proceeds through random scission of the

MATERIAL AND METHODS

Materials. Water was purified with a distillation apparatus (model WG-220, Yamato Co.) and had a conductivity of 5.5 μS·m−1. Bovine serum albumin (BSA, corn fraction V, pH 7.0, for biochemistry, Wako Chemicals) was used as a model protein in the HTW treatment experiments. BSA is a single linear polymer chain composed of 583 amino acids having a molecular weight of approximately 66 400 Da.31 Protein standards (gel filtration calibration kit, GE health care) of known molecular weights were used for the size exclusion chromatography (SEC) calibration. Protein assays were conducted with the BCA method32 using a BCA assay kit (Wako Chemicals). Calibration of total organic carbon (TOC) and total nitrogen (TN) analyses were conducted with hydroxide solution (0.1 M, Nacalai Tesque) and potassium phthalate (>99%, Wako Chemicals). Procedure. Hydrothermal Treatment of BSA with Slow Heating Rates. Tube bomb reactors (SUS316-HDF4-150, Swagelok) having an inner volume of 150 cm3 were used to study the product formation of BSA heated with water at slow heating rates. First, 80 g of BSA solution (0.33 wt %) was loaded into the reactor at room temperature. The reactor was purged with argon gas four times to remove oxygen and then the reactor was sealed. The reaction was initiated by completely immersing the reactor into a fluidized sand bath (SB160TL, ACRAFT Corp.) controlled at the reaction temperature and terminated by quenching the reactor in a cool water bath. The reaction time was defined as the time from when the reactor entered the fluidized sand bath until it was transferred into the cool water bath. The time required to achieve the reaction temperature was about 10 min. Therefore, average heating rates for the batch experiments were 0.25 K·s−1. The reaction temperature and time investigated in batch experiments were 250 °C and 10−60 min, respectively. After the reaction, the reactor was washed with 10 g of distilled water and filtered with a membrane filter (1 μm) to obtain a solid and liquid filtrate. The liquid filtrate was defined as liquid product. The obtained solid was further dried at 60 °C under vacuum until constant weight was obtained and defined as solid product. Hydrothermal Treatment of BSA with Fast Heating Rates. Fastheating-rate experiments of BSA with water were performed with the continuous-flow apparatus shown in Figure S1. Water and BSA solutions were fed through the mixing cross tee from two individual streams each pressurized by individual HPLC pumps (Figure S1). 7710

DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715

Research Article

ACS Sustainable Chemistry & Engineering main polymer chain at the peptide bonds and follows a first-order rate law giving eq 2: ln(M n,0 /M n, t ) =

∫0

t

k dt = kt

(2) −1

where t (s) is the reaction time, k (s ) is the rate constant, Mn,0 and Mn,t (Da) are the number-average molecular weights of the polymer solution obtained from reactions conducted at the given temperature and reaction times, respectively. Mean residence times were corrected for end effects as discussed in previous works.35−37 End effects occur due to inefficiency of the heating and cooling at the beginning and end of the reactor respectively, leading to differences between the reaction and mean residence time. The rate constant k was determined by fitting eq 2 to the experimental kinetic data. Batch experiments have much longer heating periods compared with the flow experiments. The decomposition of BSA may occur during the heating period before reaching the reaction temperature. Therefore, calculations were performed by taking the heating profile of the batch experiments into account and modifying the method reported by Sheehant and Savage38 according to eq 3: ln(M n,0 /M n, t ) =

∫0

t

k(t ) dt =

∫0

t

⎛ − Ea ⎞ A exp⎜ ⎟ dt ⎝ RT (t ) ⎠

(3)

where T (K) and k (s−1) are the temperature and rate constant at reaction time t (s) respectively, and Ea (J·mol−1) is the activation energy, A (s−1) is the pre-exponential factor, and R (J·mol−1·K−1) is the universal gas constant. Details on the method for determining the kinetic rate constants are given in the Supporting Information (S4).

Figure 2. Number-average molecular weight (Mn) of peptides of water-soluble products obtained for reaction of bovine serum albumin in water as a function of reaction time: (a) fast heating rate (flow experiments), (b) slow heating rate (batch experiments). Symbols: 200 °C (▲), 240 °C (⧫), 250 °C (■), 260 °C (●). Lines are fitting results.

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RESULTS AND DISCUSSION Molecular Weight Distribution of Peptide. Figure 1 shows SEC chromatograms (UV at 280 nm) of the liquid

using fast and slow heating rates. For a given temperature, the average molecular weight of the liquid products decreased as reaction time increased. As shown in Figure 2a, eq 2 was able to correlate not only the experimental data but also its trend with reaction temperature. The results in Figure 2a are strong evidence that the hydrolysis of BSA proceeded by a random scission mechanism. The kinetic fitting results at each temperature for the fast-heating-rate experiments gave kinetic rate constant values of 0.12−0.20 s−1 for temperatures from 200 to 260 °C (Table 1). The obtained kinetic values had high linearity (Figure 3) when plotting ln k versus 1/T, giving an activation energy of 16.4 kJ·mol−1 and pre-exponential factor of 8.7 s−1. The kinetic rate constant for conversion of BSA protein into amino acids is 0.0006 s−1 at 250 °C,17 which is smaller than that for conversion of BSA into peptides obtained in this work (0.17

Figure 1. Size exclusion chromatograms (UV at 280 nm) of bovine serum albumin (BSA) protein aqueous solution and BSA protein treated with water under fast and slow heating rates at 250 °C at different reaction times: (A) 0 s, (B) flow 7.27 s, (C) flow 14.6 s, (D) batch 1800 s, (E) batch 3600 s.

Table 1. Number-Average Molecular Weight (Mn) of the Liquid Product Obtained from Fast Heating of Bovine Serum Albumin Protein with Water

products obtained from the HTW treatment of BSA protein at 250 °C for fast and slow heating rates. The molecular weight distribution (MWD) shifted from high to low molecular weight substances with increasing reaction time. MWDs of the fastheating-rate experiments were narrower than those of slowheating-rate experiments. Results of the fast-heating-rate experiments imply that the BSA decomposition proceeded as a depolymerization reaction since MWD decreased without showing a pronounced change. Slow heating of BSA protein proceeded with many side reactions that formed both high- and low-molecular-weight compounds. Reaction Rate of BSA Decomposition. Figure 2a and b shows the number-average molecular weight of the liquid products obtained from the HTW treatment of BSA protein

reaction conditions temperature [°C]

reaction time [s]

Mn [Da]

k [s−1]

200

8.12 16.00 7.26 9.49 14.71 7.27 9.47 14.6 10.71 12.88 17.92

10210 ± 867 3908 ± 73 9568 ± 321 4221 ± 2 2426 ± 8 8321 ± 40 3893 ± 6 1979 ± 102 3084 ± 30 2471 ± 700 785 ± 65

0.122

240

250

260

7711

0.170

0.185

0.196

DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715

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ACS Sustainable Chemistry & Engineering

heating rate data support that BSA decomposition proceeded under a random scission mechanism. The physical structure of BSA during the hydrolysis reaction was estimated based on the change in Mn and kinetics for the fast and slow heating rates (Figure 4). For slow-heating-rate experiments, the transformation from natural BSA to aggregated BSA (via unfolded-BSA) occurs readily at low temperatures (ca. 80 °C)40 before hydrolysis takes place. Therefore, hydrolysis reactions occurred not in a random manner along the BSA polymer chain but mainly at the solid−liquid interface of the aggregated protein. The higher molecular weights obtained from slow-heating-rate experiments compared with the results of eq 3 are evidence that the decomposition of BSA was suppressed most likely due to protein aggregation (Figure 2b). These findings agree with previous studies,18,41 where the aggregation of proteins has been observed after hydrothermal treatment and the decomposition mechanism of protein conversion into amino acids has been reported based on aggregation phenomena. For fast-heating-rate experiments, much of the BSA is hydrolyzed in the unfolded state before it can form aggregate structures. In this case, water molecules are abundant around the entire protein chain and therefore the hydrolysis occurs in a random manner. In a previous study conducted using batch reactors, Powell et al.30 reported that the decomposition of BSA occurs at the ASPX and GLU-X bonds under hydrothermal conditions. In that paper,30 they explained that high reactivity of the ASP-X and GLU-X bond for hydrolysis was due to the chemical structure of the side chains both having a carboxylic acid structure. However, that proposed reaction mechanism was based on slow-heating-rate (batch) experiments, for which aggregation readily occurs.18,41 Under slow-heating-rate conditions, hydrophobic structures would appear in the aggregated solid phase and hydrophilic structures such as the carboxylic groups of ASP-X and GLU-X would appear at the solid−liquid surface, leading to selective hydrolysis reactions for ASP-X and GLU-X. Further investigation of the peptides of the liquid products focusing on the ASP-X and GLU-X is needed for understanding detailed mechanisms. Product Yields of HTW Treatment of BSA. Obtaining high protein recovery is important for industrial applications. Figure 5 and Table 2 show product yields of liquid products

Figure 3. Arrhenius plot of rate constants of bovine serum albumin decomposition to peptides [(red ■) this work] and other protein decomposition to amino acids such as (□) BSA,17 (Δ) bean dregs,20 (◊) fish proteins,19 (○) poultry.20

s−1 at 250 °C). The kinetic rate constant for conversion of proteins into amino acids from other protein sources also have smaller rate constants compared with those in this work for bean dregs being 0.0017 s−1 at 240 °C,20 fish proteins being 0.000 075 s−1 at 240 °C,19 and poultry being 0.003 35 s−1 at 230 °C39 as shown in Figure 3. Kinetic rate constants for conversion of proteins into amino acids differ greatly depend on the protein source (Figure 3), which is probably true for the case of their conversion into peptides. Figure 2b shows the experimental data obtained from slowheating-rate experiments and the projection given by eq 3. Equation 3 was found to be inapplicable for describing the time dependence of the experimental data at slow heating rates and further gave lower values than those of the experimental data. This indicates that the decomposition of BSA at slow heating rates proceeded under a suppressed mechanism compared with random scission decomposition as discussed next. Reaction Mechanism of BSA in HTW under Fast and Slow Heating Rates. Hydrolysis of proteins have been reported to depend on the hydrophilicity of the amino acids that are determined by the chemical structures of the side chains in the polymer.34 However, results in the previous section show that the decomposition rate of BSA in HTW differed greatly between experiments conducted using fast and slow heating rates. Both SEC and kinetic analysis of the fast

Figure 4. Reaction mechanisms of bovine serum albumin protein in high-temperature water under (a) fast (ca. 135−180 K·s−1, this work) and (b) slow (ca. 0.25 K·s−1) heating rates proposed in the literature.18,41 7712

DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715

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ACS Sustainable Chemistry & Engineering

the aggregation during the cooling of the reaction solution may improve the protein recovery even at short reaction times. Rogalinski et al.17 studied amino acid production in a previous study of BSA decomposition using a flow reactor at a reaction temperature of 250 °C for reaction times from 4.6 to 25.2 s. Although quantification of the peptides was not reported in that work,17 it is probable that peptides were also obtained in the liquid products. Scale-down and Scale-up. The present proposal could be applied on an analytical scale (nanogram to microgram) in a chromatographic arrangement, where a postreactor splitter would be used to reduce aqueous volume to the detector and a postcolumn separator would be used to rapidly cool reactor effluent and to remove precipitated solids prior to analysis. Increasing production from the lab scale (milligrams) to the tens-of-grams scale could be achieved by numbering-up concepts44−46 applied to the present method or with optical reactors that use photonics as the energy source.47 Productionscale (1 to 1000 kg) per year methods would require a postreactor solid−liquid separator that might be adapted from production processes for materials production reported to be on the scale of up to 1000 tons per year under hydrothermal conditions.48 Some scale-up issues for flow reactors have been reported in a previous paper by Elliott et al.49 that include slurry concentration, slurry−feed heat exchange, and postreactor solids separation; these are important factors for industrial demonstration. The present scheme targets relatively dilute (1 wt %) streams of proteins to form water-soluble highmolecular-weight peptides and minimizes byproduct formation (amino acids, ammonia) and does not use catalysts or additives; however, the use of more concentrated streams and the separation of solids after reaction to limit byproduct formation still requires additional process development for continuous operation. Sustainability Metrics. In this section, aspects of the sustainability of the fast-heating HTW process is compared with a typical enzymatic hydrolysis process for producing highmolecular-weight peptides from proteins, since enzymatic hydrolysis is highly selective. Of several types of sustainability metrics that have been proposed,50−53 the method reported by Kreuder et al.53 is the most attractive to use, because it applies a qualitative ranking scheme to each alternative process and considers all 12 principles of green chemistry that are divided into the following categories: (i) resource use, (ii) energy efficiency, and (iii) human and environmental hazards. The method of Kreuder et al.53 allows quantitative ranking among categories in considering each principle for the alternative if suitable data are available. According to analysis of available data for hydrolysis of proteins (Table S1, Supporting Information), the fast-heating rate HTW process is more favorable than the enzymatic process in the resource use and

Figure 5. Nitrogen balance of products obtained for reaction of bovine serum albumin protein in water at 250 °C as a function of reaction time: (a) fast (flow experiments) and (b) slow (batch experiments) heating rates. Symbols: (gray bars) peptides, (hatched bars) solids, (dotted bars) amino acids, (white bars) ammonia.

obtained from the hydrothermal treatment of BSA using slowand fast-heating-rate experiments conducted at 250 °C. For the batch experiments, the products were peptides of low molecular weight (500−1040 Da) and decomposition compounds such as amino acids and ammonia with no solid product. Peptide yields for slow-heating-rate experiments varied from 35 to 56%, amino acids varied from 15 to 18%, and ammonia gave yields from 14 to 30%. In previous studies, similar amino acid yields have been reported for the HTW treatment of proteins such as BSA (1.4%),17 fish proteins (7%),19 raw soybean (5%),22 raw soybean protein isolate (63%),42 raw rice bran (5%),22 bean dregs (53%),43 and defatted microalgae (12%).12 The differences of the amino acid yields are probably due to the chemical and physical properties of the starting protein that affects its solubility in water. Nevertheless, the results of this work confirm that HTW treatment of BSA using slow heating rates yields low-molecular-weight peptides, amino acids, and ammonia. For the HTW experiments conducted at fast-heating rates, the majority of the products obtained from the hydrothermal treatment of BSA were peptides of high molecular weight (8300−1470 Da) of high yields (53−84.1%), low yields of amino acids (0−0.8%), and low yields of ammonia (0−4%). The solid yields varied from 12 to 30% where short reaction times had higher values compared with those for long reaction times. Solids obtained from the reactions at fast heating rates were formed by aggregation of peptides at the cooling unit rather than during the reaction, as kinetic evaluation in previous section showed evidence that the BSA decomposition occurred under a random scission mechanism. The decrease in solid yield observed when increasing the reaction time may be due to a decrease in the molecular weight of the peptide that possibly could suppress the aggregation phenomena. Thus, suppressing

Table 2. Product Yields Obtained from Fast and Slow Heating Rates of Bovine Serum Albumin Protein with Water at 250 °C product yield [%] heating method fast (flow)

slow (batch)

reaction time [s] 7.27 9.47 14.6 600 1800 3600

peptides 52.9 64.8 84.1 72.6 49.6 45.3

± ± ± ± ± ±

0.9 3.2 3.1 7.5 4.4 2.4

amino acids 0.0 0.0 0.8 15.1 17.1 17.5

± ± ± ± ± ±

7713

0.4 0.3 0.4 0.2 0.2 0.5

ammonia

solid

± ± ± ± ± ±

30 27.4 11.5 0.0 ± 0 0.0 ± 0 0.0 ± 0

3.1 4.1 4.1 14.3 25.9 30.3

1.2 0.2 0.5 3.1 0.4 0.4

mass balance [%] 86.0 ± 68.9 ± 100 ± 3.9 103 ± 4.6 93.8 ± 95.2 ±

1.3 3.3

3.8 3.3

DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715

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ACS Sustainable Chemistry & Engineering Notes

human and environmental hazards categories, but it is less favorable than the enzymatic hydrolysis process for the energy efficiency category (Table S1, Supporting Information). However, in comparing the energy requirements of the two processes, the production of the many chemicals and enzymes required for the enzymatic hydrolysis process would have to be considered in a detailed analysis. Since the fast-heating HTW process does not require intensive separation and isolation steps and gives comparative or higher yields of high molecular weight peptides from proteins than the enzymatic hydrolysis process, it is possible that the fast-heating HTW process has lower overall energy requirements than the enzymatic hydrolysis process. Other Applications. Fast hydrolysis with HTW has been reported to achieve high biocrude yields compared with conventional high-temperature water treatment from algae biomass.38,54,55 Based on the findings in this work, it is considered that high biocrude yields obtained by fast hydrolysis are a result of random scission (hydrolysis) of the protein. That is, by fast hydrolysis, the conversion of proteins to peptides is enhanced, which could increase the population of reactive polymer chain ends (amides and carboxylic structures) to improve biocrude yields.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Next-generation Energies for Tohoku Recovery (NET) project of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. We would like to thank Dr. Masaki Kubo, Dr. Shunsuke Mochizuki, Dr. Satoshi Yamashita, Dr. Hikaru Nakazawa (Tohoku University), Dr. Iwane Suzuki, Dr. Shinya Fukuda, and Dr. Yuuhiko Tanabe (University of Tsukuba) for advice.

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CONCLUSIONS Peptides of high molecular weight can be obtained from proteins by hydrothermal treatment conducted at fast heating rates, without the addition of acid or base catalyst. The reaction mechanism for conversion of proteins into peptides in hightemperature water differs for fast and slow heating rates. For slow heating rates, protein hydrolysis occurs after protein aggregation; whereas for fast heating rates, protein hydrolysis occurs by a random scission mechanism, probably when the protein is in its denatured state before the aggregation takes place. The use of fast heating rates of water with proteins allows control of peptide molecular weight, and the technique is applicable to other protein sources not only for medical, material, and food industries but also for waste treatment and biomass refineries.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01146. S1: Details on continuous flow apparatus for hydrothermal treatment of BSA with fast heating rates (Figure S1). S2: Details on molecular weights of protein standards used for size exclusion chromatography analysis (Table S1). S3: Details on evaluation of reaction time from mean residence time for hydrothermal treatment of BSA with fast heating rates (Figure S2). S4: Details on evaluation of reaction kinetics for hydrothermal treatment of BSA with slow heating rates. S5: Sustainability metrics. (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: +81-22-795-5864. ORCID

Taku Michael Aida: 0000-0001-5382-0342 Richard Lee Smith Jr.: 0000-0002-9174-7681 7714

DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715

Research Article

ACS Sustainable Chemistry & Engineering

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DOI: 10.1021/acssuschemeng.7b01146 ACS Sustainable Chem. Eng. 2017, 5, 7709−7715