Formation of amide bond catalyzed by lipase in aqueous phase for peptide synthesis

Article abstract of DOI:10.1016/j.molcatb.2016.03.010

A dipeptide N-acetyl-l-phenylalanyl-l-tyrosinamide (N-Ac-Phe-Tyr-NH₂), with angiotensin I converting enzyme (ACE) inhibitor activity, was synthesized via porcine pancreatic lipase catalyzed amidation of N-acetyl-phenylalanine ethyl ester with l-tyrosinamide in an aqueous phase. Response surface methodology was employed to evaluate the effects of synthesis parameters. The optimum synthesis conditions obtained an 84.45% yield of N-Ac-Phe-Tyr-NH₂ with a reaction time of 3.8 min, a temperature of 20.9°C, an enzyme amount of 6.5 U, and a substrate molar ratio of 2.5:1 (Tyr:Phe). The kinetics of lipase and α-chymotrypsin catalyzed amidation was compared using the Ping-Pong mechanism. The lipase showed a lower apparent kinetic constant than α-chymotrypsin indicating that the acyl lipase intermediate had a higher affinity toward tyrosinamide in the amidation. In addition, because the lipase can avoid the secondary hydrolysis of synthesized peptide, it is expected to be an effective method for obtaining a good yield of dipeptide.

Full text of DOI:10.1016/j.molcatb.2016.03.010

Accepted Manuscript
Title: Formation of amide bond catalyzed by lipase in aqueous
phase for peptide synthesis
Author: Chia-Hung Kuo Lin Jer-An Ching-Ming Chien
Chang-Han Tsai Yung-Chuan Liu Chwen-Jen Shieh
PII:
S1381-1177(16)30049-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.03.010
MOLCAB 3344
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
14-12-2015
7-3-2016
26-3-2016
Please cite this article as: Chia-Hung Kuo, Lin Jer-An, Ching-Ming Chien, Chang-Han
Tsai, Yung-Chuan Liu, Chwen-Jen Shieh, Formation of amide bond catalyzed by lipase
in aqueous phase for peptide synthesis, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.03.010
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Formation of amide bond catalyzed by lipase in aqueous
phase for peptide synthesis
Chia-Hung Kuoa,†, Lin Jer-Anb,†, Ching-Ming Chienb, Chang-Han Tsaic, Yung-Chuan
Liuc,*, and Chwen-Jen Shieha,*
a Department of Seafood Science, National Kaohsiung Marine University,142
Haijhuan Road, Kaohsiung 81157, Taiwan
b Biotechnology Center, National Chung Hsing University, 250 Kuo-kuang Road,
Taichung 402, Taiwan
c Department of Chemical Engineering, National Chung Hsing University, 250
Kuo-Kuang Road, Taichung 402, Taiwan
† These authors contributed equally to this work.
* Corresponding Authors: Email:cjshieh@nchu.edu.tw (Chwen-Jen Shieh) ;
ycliu@dragon.nchu.edu.tw (Yung-Chuan Liu)

Graphical Abstract
1
2
3
4
5
6
7
Highlights
 Lipase catalyzed amidation for peptide synthesis.
 A high yield of dipeptide was obtained in aqueous solution.
 RSM design for optimization of reaction parameters.
 Using the kinetic model compared the catalytic efficiency.
 Kinetic constants show the advantage of lipase on amidation.
8
9
ABSTRACT:
10 A dipeptide N-acetyl-L-phenylalanyl-L-tyrosinamide (N-Ac-Phe-Tyr-NH2), with
11 angiotensin I converting enzyme (ACE) inhibitor activatity, was synthesized via
12 porcine pancreatic lipase catalyzed amidation of N-acetyl-phenylalanine ethyl ester
13 with L-tyrosinamide in an aqueous phase. Response surface methodology was

14 employed to evaluate the effects of synthesis parameters. The optimum synthesis
15 conditions obtained an 84.45% yield of N-Ac-Phe-Tyr-NH2 with a reaction time of
16 3.8 min, a temperature of 20.9 oC, an enzyme amount of 6.5 U, and a substrate molar
17 ratio of 2.5:1 (Tyr:Phe). The kinetics of lipase and α-chymotrypsin catalyzed
18 amidation was compared using the Ping-Pong mechanism. The lipase showed a lower
19 apparent kinetic constant than α-chymotrypsin indicating that the acyl lipase
20 intermediate had a higher affinity toward tyrosinamide in the amidation. In addition,
21 because the lipase can avoid the secondary hydrolysis of synthesized peptide, it is
22 expected to be an effective method for obtaining a good yield of dipeptide.
23
24
25 KEYWORDS: enzymatic peptide synthesis, amidation, porcine pancreatic lipase,
26 N-acetyl-L-phenylalanyl-L-tyrosinamide, kinetics
27
28 1. Introduction
29 Protein synthesis involves a sequence of amino acid coupling reactions to form amide
30 bonds that link amino acids in the amino-to-carboxyl direction. In the cell, protein
31 synthesis is catalyzed by the peptidyl transferase which occurs between two
32 aminoacyl-tRNAs on the ribosome [1]. However, the whole process in the cell is very

33 complex as it is mediated by more than one hundred macromolecules, including
34 mRNAs, tRNAs, ribosome, activating enzymes, and protein factors. Hence, a number
35 of enzymes have been used as practical catalysts in peptide synthesis performed in test
36 tubes [2, 3]. Enzymatic catalyzed synthesis of peptides has several advantages, such
37 as mild conditions, no racemization, more rapidly, higher yield and good
38 regioselectivity [4]. α-Chymotrypsin is the most commonly used enzyme in peptide
39 synthesis [5-7], but some peptides using lipase as catalysts have been attempted [8].
40
Lipase and α-chymotrypsin belong to the serine hydrolase family (substrate
41 enzyme intermediate formation is related to the hydroxyl group of serines at the
42 enzyme active site) [9, 10]. Lipases hydrolytically cleave ester bonds while
43 α-chymotrypsin hydrolyzes amide bonds. The mechanisms of the lipase and
44 α-chymotrypsin-catalyzed reactions are fundamentally identical. Both reactions
45 proceed via a serine hydroxyl at the enzyme active site, which attacks the carbonyl
46 carbon atom of the substrate to form an acyl enzyme intermediate [10, 11].
47 Subsequently, the acyl enzyme intermediate (acyl donor) can be deacylated by the
48 nucleophilic amino group of an amino acid substrate (acyl acceptor) to provide the
49 desired peptide, whereas deacylation with water results in hydrolysis. Hence,
50 hydrolysis and amidation pathways occur competitively [12, 13].
51 α-Chymotrypsin-catalyzed synthesis of peptides has been performed in an aqueous

52 solution [12]. Cbz-Asp-Phe-OMe has been synthesized in a monophasic
53 organic-aqueous (50% DMSO) solvent [14]. In contrast, the use of lipases to generate
54 amide bonds in organic solvents has been reported [15, 16]. Lipase catalyzed
55 aminolysis of dialkyl carbonates were run in tert-butyl alcohol [17]. Huang et al have
56 synthesized tetrapeptide Bz-Arg-Gly-Asp-Ser-NH2 using lipase in
57 dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) [18]. Recently, lipase
58 catalyzed synthesis of peptidic hydrogels in an aqueous solution has been
59 demonstrated [19]. The use of lipases to catalyze amide bond formation is an
60 interesting alternative to conventional methods using proteases, as lipases generally
61 do not cleave amide bonds that are able to avoid secondary hydrolysis of peptides. So
62 far, lipase catalyzed synthesis of peptides in an aqueous system has rarely been
63 reported.
64
Hypertension is one of the major risk factors for development of cardiovascular
65 diseases, stroke and the end stage of renal disease [20]. The Angiotensin I converting
66 enzyme (ACE) plays an important physiological role in the regulation of hypertension
67 [21]. Inhibition of ACE activity leads to a decrease in the concentration of angiotensin
68 II and consequently reduces blood pressure [22]. Several dipeptides separated from
69 garlic with tyrosine or phenylalanine residue at the C terminus have been shown to
70 inhibit ACE activity. Among these dipeptides, Phe-Tyr is the most potent ACE

71 inhibitor [23]. The antihypertensive effect of Phe-Tyr has been demonstrated in
72 spontaneously hypertensive rats where blood pressure significantly decreased after
73 oral administration [24].
74
The present work focused on lipase-catalyzed synthesis of dipeptide
75 N-Ac-Phe-Tyr-NH2 in an aqueous solution. Our purpose was to better understand the
76 solvent effect and the relationships between reaction variables (reaction time,
77 temperature, enzyme amount, and substrate molar ratio) and the response (yield of
78 N-Ac-Phe-Tyr-NH2), as well as to obtain the conditions for dipeptide
79 N-Ac-Phe-Tyr-NH2 synthesis using 5-level-4-factor composite rotatable design
80 (CCRD) and response surface methodology (RSM). In addition, a kinetic study of
81 amidation was also performed to determine the apparent kinetic parameters in order to
82 compare the specific activity and specificity of the lipase and α-chymotrypsin as
83 catalysts.
84 2. Experiment
85 2.1. Materials
86 N-acetyl-phenylalanine ethyl ester (N-Ac-Phe-OEt), L-tyrosinamide (Tyr-NH2),
87 acetonitrile (ACN), trifluoroacetic acid (TFA), Trizma base buffer (Tris buffer) and
88 porcine pancreatic lipase type II (lipase from porcine pancreas, EC 3.1.1.3, 30-90
89 Units/mg) purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents

90 and chemicals, unless otherwise noted, were of analytic grade.
91 2.2. Experimental design
92 A four-factor, five-level CCRD consisting of 27 treatments was employed in this
93 study. The manipulated (independent) variables and their respective levels selected for
94 N-Ac-Phe-Tyr-NH2 synthesis included reaction time (2–10 min), temperature (20–
95 40 oC), enzyme amount (2–10 U) and substrate molar ratio (Tyr :Phe = 1:1–3:1).
96 Table 1 shows the independent factors (xi), levels and experimental design, both
97 coded and uncoded. All reactions were carried out in duplicate.
98 2.3. Lipase catalyzed synthesis of N-Ac-Phe-Tyr-NH2
99 N-Ac-Phe-OEt (50 mM) with various amounts of L-tyrosinamide and porcine
100 pancreatic lipase were mixed with 80mM pH 9.0 Tris-HCl buffer in screw-capped
101 tubes. The reaction mixture was then placed in an orbital sharking bath (150 rpm) at
102 designed reaction temperature for the times shown in Table 1. The reaction of porcine
103 pancreatic lipase catalyzed synthesis of N-Ac-Phe-Tyr-NH2 is represented in scheme
104 1. After reaction, a 4-fold volume of termination reagent containing ACN and acetic
105 acid was added into the reaction mixture to deactivate the enzyme. Twenty microliters
106 of the reaction mixture was injected into a high-performance liquid chromatography
107 (HPLC) for composition analysis.
108 2.4. Analysis

109 The samples were analyzed by a Gilson HPLC System (Gilson 322 pump, UV-Vis
110 152 detector; L.M.I. Company) equipped with an ultraviolet detector and a Hypersil
111 ODS-2 C18 column (Thermo Instrument Systems, Runcorn, UK, 25 cm 4.6 mm). The
112 elution solvents used were 0.1% TFA of water and ACN. The flow rate was set at 1.0
113 mL/min and gradient elution was performed as follows: ACN was set at 30% for the
114 first 5 min, gradually increased to 70% between 5 and 10 min, and then returned to
115 30% for the last 5 minutes. An ultraviolet detector was set at a wave-length of 254 nm.
116 The yields were calculated from the peak areas of the substrate and dipeptide.
117 2.5. Statistical analysis
118
Using SAS software, the experimental data (Table 1) were then analyzed by the
119 response surface regression (RSREG) procedure to fit the following second-order
120 polynomial Eq. 1:
121
122 where Y is the yield of N-Ac-Phe-Tyr-NH2; β0, βi, βij, are constant coefficients; xi and
123 xj are uncoded independent variables. The option of ridge max option in the SAS
124 software was employed to compute the estimated ridge of maximum response for
125 increasing the radius from the center of the original design.
126 2.6. Kinetic study
127 To determine the kinetics of the amidation, reaction mixtures were prepared by a fixed

128 N-Ac-Phe-OEt concentration (10 mM) combined with various L-tyrosinamide
129 concentrations (5-25 mM). The amount of enzyme was 55 U in Tris buffer at 20 oC
130 for 1min. Initial reaction rates were expressed as mM produced N-Ac-Phe-Tyr-NH2
131 per min.
132 3. Results and discussion
133 3.1. Solvent selection for synthesis of N-Ac-Phe-Tyr-NH2
134 The substrates, N-Ac-Phe-OEt and Tyr-NH2, have a reasonable solubility in the
135 aqueous Tris-HCL buffer (80mM, pH=9) and also in the organic solvents: ACN and
136 DMSO. To understand the relation of the solvent effects on the lipase catalyzed
137 synthesis of N-Ac-Phe-Tyr-NH2, these solvents were employed to investigate the
138 effects of solvent on the dipeptide yield. The solvent compositions and the resulting
139 N-Ac-Phe-Tyr-NH2 yields from the tests are listed in Table 2. In the single solvent
140 environments, the 60.7% yield of N-Ac-Phe-Tyr-NH2 was obtained in a Tris-HCL
141 buffer. However, the dipeptide was not synthesized in pure ACN or DMSO solvent.
142 In addition, the yield of N-Ac-Phe-Tyr-NH2 in aqueous-ACN and aqueous-DMSO
143 was decreased to 12.6 and 40.6%, respectively. As the pK2 of tyrosine is at 9.1, this
144 means that ½ of the amino acid molecules have lost the H+ from the NH3 group at pH
145 9. Therefore, the α-amino group of Tyr-NH2 contains the nitrogen lone pair that
146 attacks the carbonyl carbon atom of N-Ac-Phe-OEt-enzyme intermediate to form an

147 amide bond. These results indicate that the aqueous Tri-HCL buffer at pH 9 was
148 required for deacylation of acyl-enzyme intermediate with a nucleophile reactant
149 (Tyr-NH2).
150 3.2. Model fitting
151 The RSM combined with a four-factor/five-level CCRD employed in this study was
152 more efficient in reducing the experimental runs and time needed to investigate the
153 relationship between the synthesis conditions and dipeptide yield. The RSREG
154 procedure of SAS was employed to fit the second-order polynomial equation (1) to
155 the experimental data-percent mole conversions (Table 1). Among the various
156 treatments, the greatest mole conversion (83.6%) was treatment 11 (4 min, 25oC,
157 enzyme amount 4 U, substrate mole ratio 2.5:1), and the smallest conversion (only
158 60.5%) was treatment 18 (8 min, 35 oC , enzyme amount 8 U, substrate mole ratio
159 1.5:1). From the SAS output of RSREG, the second-order polynomial equation (2) is
160 given below:
161
162
163
164
(2)
165 The analysis of variance (ANOVA) results indicate that this second-order polynomial

166 model was highly significant and adequately represents the actual relationship
167 between the response (percentage molar conversion) and the significant variables with
168 very a small p value (0.0001) and a satisfactory coefficient of determination (R2 =
169 0.9744). Furthermore, the overall effect of the four synthesis variables on the
170 percentage molar conversion was analyzed further by a joint test. The results reported
171 in Table 3 reveal that temperature (X2) and substrate molar ratio (X4) are the most
172 important factors, exerting a statistically significant overall effect (p < 0.0001) on the
173 response molar conversion.
174 3.3.Mutual effect of parameters
175 The substrate molar ratio and temperature were investigated in the ranges 1:1–3:1 and
176 20-40 oC, respectively. Figure 1 shows the effect of varying the substrate molar ratio,
177 temperature and their mutual interaction on N-Ac-Phe-Tyr-NH2 synthesis in 4 min
178 using a constant enzyme amount of 4 U. The lowest molar conversion was 60% at the
179 lowest substrate molar ratio of 1:1 and at the highest temperature of 40 oC. A reaction
180 condition at substrate molar ratio of 2.5:1 and a temperature of 20 oC obtained ~ 85 %
181 mole conversion. These results indicate that both substrate molar ratio and
182 temperature were important parameters. In the N-Ac-Phe-Tyr-NH2 synthesis reaction,
183 L-tyrosinamide was competed with water to react with acyl enzyme intermediate.
184 Therefore, overloading of L-tyrosinamide benefitted the reaction process in forming

185 an amidation product and in quicker completion. Figure 2 represents the effect of
186 varying the substrate molar ratio and reaction time on synthesis, with a constant
187 enzyme activity of 2 U at 20 oC. At any substrate molar ratio, the molar conversion
188 was not affected significantly by reaction time. As shown in the results contained in
189 Table 3, that reaction time (X1) has a less significant effect (p > 0.05) on the synthesis
190 of N-Ac-Phe-Tyr-NH2. The relationships between the reaction factors and the
191 response can be better understood by examining the planned series of contour plots
192 (Figure 3). These were generated from the predicted model (Eq. 2) by holding both
193 the substrate molar ratio (1:1, 2:1, or 3:1) and the enzyme amount (2, 6, or 10 U)
194 constant. Figure 3 A, B and C represent the same substrate molar ratio (1:1); and A, D
195 and G represent the same enzyme activity (2 U). Such an application could be adopted
196 to study the synthesis variables simultaneously in a five-dimensional space. In general,
197 all nine contour plots shown in Fig. 3 exhibit similar behaviors; the predicted molar
198 conversion increased with an increasing substrate molar ratio, whereas the molar
199 conversion decreased with corresponding increases in temperature. Therefore,
200 temperature (X2) and substrate molar ratio (X4) were the most important variables for
201 N-Ac-Phe-Tyr-NH2 synthesis, with small p values (Table 3), and can be considered as
202 indicators of effectiveness and economic performance.
203 3.4. Optimum synthesis conditions

204 The optimum conditions were determined by ridge max analysis. This method
205 computes the estimated ridge of maximum response for increasing radii from the
206 center of original design. The ridge max analysis (Table 4) was verified that the
207 maximum molar conversion of N-Ac-Phe-Tyr-NH2 dipeptide was 85.0±1.6% at a
208 reaction time of 3.8 min, a temperature of 20.9 oC, a substrate molar ratio of 2.53:1
209 and an enzyme amount of 6.5 U. The validity of the predicted model was tested by
210 repeating the experiments three times. At the suggested optimal synthesis condition,
211 the actual molar conversion of N-Ac-Phe-OEt was 84.40±0.98%. The results indicated
212 that the observed value was almost identical to the predicted value.
213 3.5. Kinetic study
214 α-Chymotrypsin is most used enzyme in peptide synthesis. In order to compare the
215 catalytic efficiency of lipase and α-chymotrypsin in N-Ac-Phe-Tyr-NH2 synthesis, the
216 kinetic constants were used as an indicator in this study. Most of lipases catalyzed
217 transesterification has been proposed to follow the Ping-Pong Bi–Bi mechanism [25,
218 26], supposing the initial amount of products to be zero with no inhibition of either
219 substrates or products. The general Ping-Pong Bi–Bi mechanism rate equation derived
220 by Albert is given as [27]:
221
(3)
222 where vO is the initial reaction rate, Vm is the maximum rate of reaction, [A0] is the

223 initial concentration of N-Ac-Phe-OEt, [B0] is the initial concentration of
224 tyrosinamide, KAm is the Michaelis constant for N-Ac-Phe-OEt, and KBm is the
225 Michaelis constant for tyrosinamide. At constant but not saturating [A0], the equation
226 can be rearranged as Eq. 4, which is the form of the Michaelis–Menten equation.
227
(4)
228 where
;
and
229 In previous study, the α-chymotrypsin catalyzed synthesized dipeptide using the
230 substrate concentrate of 10 mM N-Ac-Phe-OEt and enzyme amount of 71.5 U [7]. In
231 order to compare the apparent kinetic constants of lipase and α-chymotrypsin, the
232 N-Ac-Phe-OEt concentration [A0] was fixed at 10 mM with various tyrosinamide
233 concentrations (5-25 mM) using an enzyme amount of 55 U. At the sufficient enzyme
234 amount situation, increase in enzyme amount increases the reaction rate, but the final
235 molar conversion doesn't increase significantly [28-29]. The Lineweaver–Burk plot of
236 the reciprocal initial rate versus the reciprocal concentration of tyrosinamide was
237 linear (Figure 4). The curve shows good linearity even at a high tyrosinamide (25 mM)
238 concentration; this indicates that no tyrosinamide inhibition occurred. In addition, the
239 initial rates were determined by the tyrosinamide concentration [B0], which was fixed
240 at 10 mM with various N-Ac-Phe-OEt concentrations (5-15 mM). That the initial rate
241 increased with increasing N-Ac-Phe-OEt concentrations indicates no N-Ac-Phe-OEt

242 inhibition occurred, so Eqs. (3) and (4) can be applied in this study. According to the
243 Ping-Pong Bi–Bi mechanism, the ester substrate N-Ac-Phe-OEt (acyl donor) first
244 combines with enzyme to form a N-Ac-Phe-OEt–enzyme complex, which then
245 intermediately transfers to acyl (N-Ac-Phe) enzyme intermediate and releases ethanol.
246 The acyl enzyme intermediate can then be deacylated by the nucleophilic
247 tyrosinamide (nucleophile) to yield N-Ac-Phe-Tyr-NH2 and free enzyme is released.
248 The values of apparent kinetic constant (K2), apparent V'm, and V'm/K2 were 7.74 mM,
249 13.14 mM/min, and 1.69 min for lipase and 11.56 mM, 20.24 mM/min, and 1.75 min
250 for α-chymotrypsin. The V'm is directly proportional to K1, and the KAm is inversely
251 proportional to K1. As compared with lipase, α-chymotrypsin exhibits a higher V'm
252 value, representing its KAm value is less than that of lipase. A lower KAm value
253 indicates a higher affinity of α-chymotrypsin for N-Ac-Phe-OEt. This is probably due
254 to phenylalanine is one of specific amino acid for α-chymotrypsin. On the other hand,
255 the value of the apparent kinetic constant K2 is also a measure of the strength of the
256 N-Ac-Phe-OEt–enzyme complex; the lower value of K2 in Eq. 4, indicates the higher
257 affinity of acyl enzyme intermediate for tyrosinamide. As compared with
258 α-chymotrypsin, lipase exhibits a lower K2 value, thus indicating a higher affinity of
259 acyl lipase intermediate for tyrosinamide. The V'm/K2 is often considered to be a
260 suitable parameter to represent enzyme performance [30]. The apparent kinetic

261 constant V'm/K2 ratio of lipase and α-chymotrypsin were almost the same indicating
262 that the catalytic efficiency is quite close, but the use of lipase in peptide synthesis can
263 avoid secondary hydrolysis of peptides.
264 4. Conclusions
265 In this study, the optimal conditions for lipase catalyzed synthesis of
266 N-Ac-Phe-Tyr-NH2 was successfully assessed and demonstrated. The pH plays an
267 important role in peptide synthesis in the aqueous phase. The dipeptide molar
268 conversion can be further enhanced by the central composite design and response
269 surface methodology. A RSM model was obtained to describe the relationship
270 between the molar conversion of N-Ac-Phe-Tyr-NH2 and the four parameters of
271 reaction time, temperature, enzyme amount and substrate molar ratio. The results
272 indicated the temperature and substrate molar ratio significantly affected the
273 enzymatic synthesis. An optimal conversion of N-Ac-Phe-Tyr-NH2 (around 85%) was
o
274 observed at a reaction time of 3.8 min, a reaction temperature of 20.9 C, a substrate
275 molar ratio (Tyr:Phe) of 2.53:1 and an enzyme amount of 6.5 U. In the kinetic study,
276 lipase shows a similar catalytic efficiency as α-chymotrypsin in N-Ac-Phe-Tyr-NH2
277 synthesis. The present lipase catalyzed amidation procedure offers some important
278 advantages over the chemical methods or proteases for application in industrial
279 processes.

280
281 Acknowledgements
282 This work was supported by research funding grants provided by the Ministry of
283 Science and Technology of Taiwan (MOST 104-2221-E-005-061-MY3 and MOST
284 104-2218-E-022-001-MY2) and was also supported in part by the Ministry of
285 Education, Taiwan, under the ATU plan.
286
287

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337 Figure captions:
338 Scheme 1. Enzymatic synthesis of N-Ac-Phe-Tyr-NH2 by porcine pancreatic lipase.
339 Figure 1. Response surface plots showing the effect of substrate molar ratio,
340 temperature and their mutual interaction on N-Ac-Phe-Tyr-NH2 synthesis.
341 Figure 2. Response surface plots showing the effect of substrate molar ratio,
342 reaction time and their mutual interaction on N-Ac-Phe-Tyr-NH2 synthesis.
343 Figure 3. Contour plots of yields for N-Ac-Phe-Tyr-NH2. The number associated
344 with the plots indicates the predicted yield at the given reaction condition. Numbers
345 within contour plots indicate molar conversions at given reaction conditions.
346 SR, substrate molar ratio (Tyr:Phe).
347 Figure 4. Lineweaver–Burk plot of reciprocal initial reaction rate versus reciprocal
348 tyrosinamide (S0) concentrations at fixed N-Ac-Phe-OEt concentration.
349
350

351 Table 1. Four-factor, five-level CCRD and experimental results of dipeptide
352 derivative yield in response surface analysis.
Time
(min)
Temperature
(oC)
Enzyme
activity
(U)
Substrate
Molar ratio
(Tyr/Phe)
X4
Yieldb
(%)
Treatment
NO.a
X1
X2
X3
Y
1
0 (6)c
0 (6)
0 (6)
0 (6)
-1 (4)
1 (8)
-1 (4)
-1 (4)
0 (6)
0 (6)
-1 (4)
0 (6)
1 (8)
1 (8)
1 (8)
-1 (4)
-1 (4)
1 (8)
-2 (2)
-1 (4)
1 (8)
0 (6)
0 (6)
-1 (4)
1 (8)
2 (10)
1 (8)
0 (30)
-2 (20)
0 (30)
0 (30)
1 (35)
1 (35)
-1 (25)
1 (35)
0 (30)
2 (40)
-1 (25)
0 (30)
-1 (25)
-1 (25)
1 (35)
1 (35)
1 (35)
1 (35)
0 (30)
-1 (25)
-1 (25)
0 (30)
0 (30)
-1 (25)
-1 (25)
0 (30)
1 (35)
0 (6)
0 (6)
-2 (2)
2 (10)
1 (8)
-1 (4)
1 (8)
-1 (4)
0 (6)
0 (6)
-1 (4)
0 (6)
-1 (4)
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
0 (6)
-1 (4)
-1 (4)
0 (6)
0 (6)
1 (8)
1 (8)
0 (6)
1 (8)
0 (2)
75.3±0.32
81.5±1.25
77.4±0.86
72.4±2.36
66.4±1.89
67.6±2.01
72.2±3.06
68.3±0.99
57.7±1.54
68.4±0.54
83.6±0.69
75.9±0.21
74.1±0.13
69.6±0.88
77.2±2.16
77.0±2.81
77.9±1.67
60.5±1.53
77.3±1.49
75.6±0.12
79.2±0.22
81.7±0.88
73.2±0.54
81.4±1.64
79.8±0.32
73.8±0.75
72.6±1.78
2
0 (2)
3
0 (2)
4
0 (2)
5
-1 (1.5)
-1 (1.5)
-1 (1.5)
-1 (1.5)
-2 (1)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
0 (2)
1 (2.5)
0 (2)
-1 (1.5)
-1 (1.5)
1 (2.5)
1 (2.5)
1 (2.5)
-1 (1.5)
0 (2)
-1 (1.5)
1 (2.5)
2 (3)
0 (2)
1 (2.5)
1 (2.5)
0 (2)
1 (2.5)
353 a The treatments were run in random order.
354 b Yield was the average (
) of duplicated experiments.
355

356 Table 2. Solvent effect on the peptide (N-Ac-Phe-Tyr-NH2) yield from
357 N-Ac-Phe-OEt and Tyr-NH2.
Yield
Solvent Component(s)
Peptide (%)
Tris-HCL buffer
60.7
—
ACN
DMSO
—
Tris-HCL : ACN( 1:1)
Tris-HCL : DMSO (1:1)
ACN:DMSO (1:1)
12.6
40.6
—
358 — No detected for HPLC analysis.
359

360 Table 3. Analysis of variance for joint test for dipeptide derivative.
Factor
Degrees of freedom Sum of squares
Prob.>Fa
0.0334
Reaction Time(X1)
Temperature(X2)
5
5
5
5
38.952315
236.735648
59.029537
668.997315
<0.0001
0.0079
Enzyme amount (X3)
Substrate molar ratio(X4)
<0.0001
361 aProb. > F, level of significance.
362

363 Table 4. Estimated ridge of maximum response for variable percentages of molar
364 production.
Estimated
Response
Actual
Coded
Response
X1
(min)
5. 9
5.6
X2
X3
(U)
5.9
5.7
5.8
6.5
X4
(Tyr/Phe)
2.07
Radius (% conversion) (% conversion)
(oC)
29.5
27.6
24.2
20.9
0.1
0.4
0.8
1.2
76.0±0.84
79.1±0.77
82.2±0.83
85.0±1.60
79.6±1.01
80.4±1.43
81.4±0.81
84.4±0.98
2.29
4.8
2.46
3.8
2.53
365
366

367
368
369
370
Scheme 1. Enzymatic synthesis of N-Ac-Phe-Tyr-NH2 by porcine pancreatic
371
372
lipase.

373
374
90
85
80
75
70
65
60
55
3.0
2.5
2.0
35
1.5
30
25
1.0
20
375
376
Figure 1. Response surface plots showing the effect of substrate molar ratio,
temperature and their mutual interaction on N-Ac-Phe-Tyr-NH2 synthesis.
377
378

379
380
88
86
84
82
80
78
76
74
72
70
e)
3.0
/Ph
2.5
Tyr
(
o
ati
r
2.0
lar
o
9
8
1.5
7
6
ate m
5
str
4
b
1.0
3
u
2
S
381
382
Figure 2. Response surface plots showing the effect of substrate molar ratio,
reaction time and their mutual interaction on N-Ac-Phe-Tyr-NH2 synthesis.
383
384

385
386
(A)
(B)
(C)
Enzyme 10 U
Enzyme 6 U
Enzyme 2 U
387
388
(D)
(E)
(F)
389
390
(G)
(H)
(I)
391
392
393
Reaction Time (min)
394 Figure 3. Contour plots of yields for N-Ac-Phe-Tyr-NH2. The number associated
395 with the plots indicates the predicted yield at the given reaction condition. Numbers
396 within contour plots indicate molar conversions at given reaction conditions.
397 SR, substrate molar ratio (Tyr:Phe).
398

399
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
y = 0.5888x + 0.0761
R2 = 0.9934
y = 0.571x + 0.0494
R2 = 0.9903
α -chymotrypsin
Lipase
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
1/[S0]
400
401
Figure 4. Lineweaver–Burk plot of reciprocal initial reaction rate versus reciprocal
tyrosinamide (S0) concentrations at fixed N-Ac-Phe-OEt concentration.
402
403
404