SDS-PAGE and Native-PAGE analysis - 溶藻弧菌胺醯組胺酸雙胜肽酶之生化特性分析及其功能性胺基酸之研究

Chapter 2 Methods

2.4 SDS-PAGE and Native-PAGE analysis

After expression and purification, gel electrophoresis was used to check the expression level, protein purity, and determination of the molecular weight. The samples were electrophoresed on a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Table 3). Each 10 μL sample was mixed with 2 μL 5X SDS-PAGE sample buffer and incubated at 95 ºC for 5 min to denature proteins. The electrophoresis was performed with 1X SDS-PAGE running buffer at 90 Volt for 30 min following 120 Volt for 1.5 hrs. The SDS-PAGE was stained with stain buffer containing Coomassie Brilliant blue

R-250 for 30 min and destained with destain buffer I (methanol/acetic acid/water = 4:1:5, v/v/v) for 20 min and following destain buffer II (methanol/acetic acid/water = 1.2:0.05:8.75) overnight.

Native-PAGE was performed to check the native form of PepD. The purified and dialyzed proteins fractions were electrophoresed on a 7.5% Native-PAGE (Table. 3). The experimental steps were similar to SDS-PAGE analysis besides the gel containing no SDS and without denaturing treatment. Each 10 μL sample was mixed with 2 μL 5X Native-PAGE sample buffer and was performed immediately with iced 1X Native-PAGE running buffer at 90 Volt for 3 hrs with 4 ºC circulating water bath. The proteins were stained and destained in the same way as SDS-PAGE analysis.

Table 3. Solutions and volumes for preparation of SDS-PAGE and Native-PAGE separating gel and stacking gel.

Separating gel Stacking gel 7.5% 10% 12% 12.5% 15% 20% 4%

ddH₂O (mL) 16.8 13.88 11.55 11 8.05 2.22 1.7 1.5 M Tris-HCl, pH 8.8

(mL) 8.75 8.75 8.75 8.75 8.75 8.75 -

1 M Tris-HCl, pH 6.8 (mL) - - - 1.25

10 % SDS (mL)^a 0.35 0.35 0.35 0.35 0.35 0.35 0.1

30% acrylamide / 1%

N,N’-methylenediacrylamid e (mL)

8.75 11.67 14 14.6 17.5 23.33 0.01

TEMED (mL) 0.028 0.014 0.014 0.014 0.014 0.014 6.8

10 % Ammonium persulfate

(APS)^b (mL) 0.35 0.35 0.35 0.35 0.35 0.35 10

Total (mL) 35 35 35 35 35 35 10

a Replace SDS with ddH2O when preparing Native-PAGE

bRecommended to prepare freshly and mix at last

2.5 Western blotting analysis

After gel electrophoresis, the resultant Native-PAGE and a nitrocellulose (NC) membrane were soaked instantly in the transfer buffer. Following transferred the protein immediately to an NC membrane with a blotting apparatus at 90 Volt for 1 hr, the NC membrane was blocked with blocking buffer for 1 hr at RT. The membrane was then washed 3 times with 1X PBS buffer and incubated with the primary anti-PepD monoclonal antibody (mAb) at 1:1,000 dilutions with 1X PBS buffer for 1 hr at RT with gentle shaking, followed by washed 5 times with 1X PBST buffer to remove the unbound primary antibodies. The washed membrane was further incubated with the goat anti-mouse IgG conjugated HRP at 1:5,000 dilutions with 1X PBS buffer for 1 hr at RT with gentle shaking. Finally, the membrane was washed with 1X PBST buffer for 5 times. The immunoreactive bands were visualized with a chemiluminescence reagent and the autoradiography film.

2.6 Enzymatic activity assay of Vibrio alginolyticus PepD

The PepD activity was determined according to Teufel et al.⁴² on the basis of measurement of histidine by using of the o-phthalaldehyde (OPA) reagent. The substrate L-carnosine (β-Ala-L-His) would be hydrolyzed to β-Alanine and L-Histidine. The fluorescence of the derivative of histidine with OPA was detected at λEx: 355 nm and λEm: 460 nm.

There were 20 μL purified enzyme (0.5 mg/mL) and 80 μL 50 mM Tris-HCl pH 6.8 buffer reacted with 0.5 mM L-carnosine for 20 min. Liberated histidine was derivatived by adding 100 μL OPA reagent and incubated at 37 ºC in darkness for 5 min. The reaction containing only buffer with L-histidine and L-carnosine reacted with OPA were served as positive and negative control, respectively. All reactions were carried out in triplicate.

Fluorescence of the histidine derivatived with OPA was measured by Fluoroskan Ascent FL.

(λEx: 355 nm and λEm: 460 nm).

2.7 Substrate specificty of Vibrio alginolyticus PepD

To investigate the substrate specificity of PepD, various Xaa-His dipeptides, including β-Ala-L-His (L-carnosine), α-Ala-L-His, Gly-His, Val-His, Leu-His, Ile-His, Tyr-His, Ser-His, His-His, β-Asp-L-His, and γ-Amino-butyryl-His (GABA-His, homocarnosine) and two histidine-containing tripeptides, Gly-Gly-His and Gly-His-Gly, were used. The activity on

L-carnosine was defined as 100%. The enzymatic activity analysis method and reaction condition were as described on 2.6.

2.8 Enzyme Kinetics

For determination of Vmax, Km, and kcat of V. alginolyticus PepD and compared the hydrolysis efficiency with the wild-type and mutant PepD, the method described by Csámpai et al.⁵⁹ was modified to use by using High Performance Liquid Chromatography (HPLC) with Fluorescence Detector (FLD). The system, which consists of Agilent 1100 Series Quaternary pump, Autosampler, Fluorescence Detector and Inertsil ODS-3 (7 μm, 7.6 mm×250 mm) column, was used. The eluent system consisted of two components: eluent A was 0.05 M sodium acetate of pH 7.2, while eluent B was prepared from 0.1 M sodium acetate–acetonitrile–methanol (46:44:10, v/v/v) (titrated with glacial acetic acid or 1 M sodium hydroxide to pH 7.2). The gradient program was as described on Table 4. The fluent

flow-rate was 0.8 mL/min at 30 ºC.

Table 4. The fluent gradient program

Step Time (min) A (%) B (%) 1 0 100 0 2 5 50 50 3 15 0 100 4 25 0 100

Different concentrations of L-carnosine (1, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, 0.005, and 0.0025 mM) were added as substrates to initiate enzymatic reactions. After 20 min incubation at 37 ºC, the samples were mixed with OPA reagent for 5 min incubation at 37 ºC then injected by autosampler. Fluorescence of the histidine with derivatived OPA was measured by FLD (λExc: 355 nm and λEm: 460 nm). Various concentration of L-histidine solution (0.05, 0.025, 0.01, 0.005, 0.0025, 0.001, 0.0005, 0.00025, and 0.0001 mM) derivatived with OPA reagent were detected as method described above to serve as standards.

2.9 Site-directed mutagenesis on Vibrio alginolyticus pepD

Site-directed mutagenesis was performed by using the QuickChange site-directed mutagenesis kit to create the mutants. Mutagenic primers were designed and pET-28a(+)-pepD plasmid (wild-type) was used as the template: the PCR reaction was carried out by using the nonstrand-displacing action of pfuTurbo DNA polymerase to extend and incorporate the mutagenic primers (Appendix 1.), and resulting in the nicked circular strands. The PCR mutagenesis reaction was performed in the 96-well GeneAmp^® PCR System 9700 Thermal Cycler as recommended by the manufacturer of PfuUltra^TM High-Fidelity DNA polymerase. Each reaction added 100 ng of wild-type plasmid, 5 μL 10X Pfu polymerase buffer, 4 μL 2.5 mM dNTP mix, 1 μL of each 12.5 μM primer, 1 μL (2.5 U) Pfu polymerase and ddH2O to the final volume of 50 μL (Table 5). The PCR products with

wild-type and mutant plasmids were incubated with DpnI for 4 hrs at 37 ºC to selectively digest the methylated, non-mutated parental wild-type plasmids. After DpnⅠdigestion, the mutant plasmid was transformed into E. coli. XL1-Blue competent cells, with selection for kanamycin resistance. After the successful mutagenesis confirmed by restriction enzymes and DNA sequencing of plasmid, the desired mutant plasmids were transformed into E. coli BL21( DE3 ) pLysS competent cells for expression of the mutant pepD proteins.

Table 5. Reaction conditions and cycling parameters for the PCR mutagenesis reaction Segment Cycles Temperature Time

1 1 95 ºC 2 minutes

pET-28a(+)-pepD plasmid 0.5 Pfu polymerase (2.5U/μL) 1 10X Pfu polymerase buffer 5 Primer 1 (12.5 μM) 1 Primer 2 (12.5 μM) 1 dNTP mix (2.5 mM each) 4

ddH2O 37.5

Total 50 μL

2.10 Circular dichroism (CD) spectroscopy

The secondary structure of the wild-type and the mutant pepD proteins were confirmed by monitoring CD spectra. The protein sample concentration was 0.2 mg/mL in 50 mM Tris-HCl, pH 6.8 buffer. The CD spectra were recorded every 1 nm between 200 to 300 nm wavelength used a quartz cuvette of 1 mm path-length in a Jasco J-715 spectropolarimeter, Only 50 mM Tris-HCl, pH 6.8 buffer was as the control. The results were scanned 4 times and averaged. Converted the data into mean residue ellipticity (MRE) by using the equation :

［θ］MRE = (MRW × θobs/c × d).⁶⁰ θobs is the observed ellipticity (in millidegrees) at the respective wavelength, MRW is the mean residue of the enzyme (MRW = M/n, M = 53548.8 g/mole, n = 490 amino acid residues), d is the cuvette path-length in cm, and c is the protein concentration in mg/mL.⁶⁰

2.11 Crystallization

Purified recombinant PepD was produced as previously described on 2.2. The purified enzyme was concentrated to 10 mg/mL and dialysed to against Tris-HCl buffer with 20 mM HEPES buffer by Centricon YM30. Using the hanging drop technique, one small droplet of the sample mixed with crystallization reagent was dropped on a

siliconized glass cover slide, and the cover slide would invert to over the reservoir in vapor equilibration with the reagent. In this experiment, hanging drops were formed by mixing 1 μL enzyme solution with 1 μL of crystallization reagent at 20 ºC with the reservoir solution.

2.12 Analytical Sedimentation Velocity Ultracentrifugation

Sedimentation velocity is an analytical ultracentrifugation (AUC) method that measures the molecular moved rate for providing both the molecular mass and the shape of molecules.⁶¹ This technique can distinguish the native state of the protein in either a monomer, dimmer, or even tetramer form. The data were evaluated according to the g*(s) method developed by Walter Stafford.⁶² Since the g*(s) analysis yields both the sedimentation coefficient s from the peak of the curve, the apparent molecular weight can also be determined. Depending on the application and optical system, the protein concentration ranging 0.1 mg/mL to 0.5 mg/mL was used and the sample volume was about 500 μL. Sample was equilibrated with 20 mM Tris-HCl pH 6.8 buffer and this equilibrated buffer was used as another reference control into the reference sector. The sedimentation velocity analysis was performed at National Tsing Hua University.

Chapter 3 Results

3.1 Expression and Purification of Vibrio alginolyticus PepD

Vibrio alginolyticus PepD was sucessfully expressed in E. coli BL21(DE3)pLysS and purified by Ni-NTA column chromatography. The Ni-NTA resin-bound PepD would be eluted with high purity by 20 mM Tris-HCl, 0.5 M NaCl, pH 6.8 buffer containing 150 mM imidazole. The pure elutent fraction was collected and dialyzed with 20 mM Tris-HCl, pH 6.8 buffer at 4 ºC to remove the salts. The dialyzed protein on SDS-PAGE revealed a single band with molecular mass of approximately 55 kDa (Fig. 5), quite close to the calculated molecular mass, 53.6 kDa, of Vibrio alginolyticus PepD. The purified Vibrio alginolyticus PepD was also confirmed by Western blotting with an anti-PepD mAb (Fig. 5).

M 1 2 3 4

ig. 5 SDS-PAGE and Western blot analysis of purified PepD

f E.coli BL21(DE3)pLysS

The concentration of purified PepD was determined by BCA Protein Assay Reagent and in total 5 mg pure PepD from

30 66 45

(kDa)

(a) Lane M:LMW protein marker;Lane 1:cell crude extracts o

carrying pET-28a(+); Lane 2:cell crude extracts of E.coli BL21(DE3)pLysS carrying pET-28a(+)-pepD; Lane 3:purified PepD from Ni-NTA column; Lane 4:western blotting analysis of purified PepD with anti-PepD mAbs

300 mL E. coli cells could be abtained. The purified PepD was further characterized with the activity assay as described in 2.6.

3.2 Enzymatic activity assay

As described in 2.6, the purified wild-type PepD was subjected to the activity assay with L-c

.3 Substrate Specificity of Vibrio alginolyticus PepD

ase with broad substrate spe

arnosine as a substrate, which would be hydrolyzed to β-alanine and L- histidine.

According to Teufel et al.⁴² on the basis of measurement of histidine by use of o-phthalaldehyde (OPA) reagent. The histidine-OPA derivative was detected at λEx: 355nm and λEm: 460nm. The purified PepD was confirmed as a member of carnosine-hydrolyzing enzymes capable of catalyzing the hydrolysis of L-carnosine to β-alanine and L-histidine which further producing the detectable fluorescence derivative of L-histidine while reacting with OPA reagent in 50 mM Tris-HCl, pH 6.8 buffer.

3

The PepD from E. coli has been identified as a dipeptid

cificity.³⁰ The substrate specificity of PepD from Vibrio alginolyticus was determined with eleven Xaa-His dipeptides, two non Xaa-His dipeptides, and two His-containing tripepides, and compared with the data from E. coli. The experimental method was as described in 2.7. The enzyme activity on L-carnosine (β-Ala-L-His), the known substrate of aminoacylhistidine dipeptidase (PepD), was defined as 100% (Fig. 6). The highest enzyme activity was observed from the hydrolysis of His-His, which was about two times higher than that of the L-carnosine. Moreover, the hydrolysis of α-Ala-L-His, which only differs in the orientation of the alanine also showed 1.5 times higher activity than L-carnosine hydrolysis.

The relative dipeptidase activities with the other Xaa-His dipeptides substrates including Val-His, Leu-His, Tyr-His, Ile-His and Ser-His were also superior to that of carnosine degradation, and the enzyme could also hydrolyze Gly-His with good activity. The enzyme showed no apparent activity toward β-Asp-L-His and γ-Amino-butyryl-His (GABA-His, homocarnosine). In addition, the non-Xaa-His dipeptides including His-Ile, His-Val, as well as tripeptides containing histidine in the central or C-terminal position were not degraded,

indicating that V. alginolyticus PepD is a dipeptidase in activity.

Fig. 6 Substrate specificity of PepD for Xaa-His dipeptides and histidine-containing

.4 Enzyme Kinetics of Vibrio alginolyticus PepD

-carnosine was performed as desc

tripeptides. Purified recombinant PepD proteins were incubated for 25 min at 37 ºC with 11 Xaa-His dipeptides, 2 non Xaa-His dipeptides and 2 His-containing tripepides, and the activity was measured as standard activity assay (see 2.6). Values are expressed as relative activity setting the degradation of carnosine to 100%

3

The enzyme kinetics of V. alginolyticus PepD for L

ribed in 2.8. The Vmax and Km values of V. alginolyticus PepD (2 μg, 0.186 μM) for

L-carnosine calculated from the respective Lineweaver-Burk plot were 1.6 μM/min and 0.36 mM, respectively (Fig. 7). Therefore, the turnover number (kcat, kcat = Vmax/[E]T) of V.

alginolyticus PepD for L-carnosine in 50 mM Tris-HCl, pH 6.8 at 37 ºC was 8.6 min^-1 and the catalytic efficiency (kcat/Km) was 0.398 mM^-1s^-1. The determined Km value of PepD was 5.64 mM from E. coli³⁰ and 0.25 mM from S. typhimurium³⁴. The other kinetics values including

kcat, kcat/Km, and specific activity of PepD, however, were first identified.

(a)

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0 0.5 1 1

[L-carnosine] (mM) V0 (uM/min)

(b)

y = 0.2149x + 0.6028 R² = 0.9865

0 5 10 15 20 25

0 20 40 60 80 100 120

1/[S] (1/mM) 1/V0 (min/uM)

Fig. 7 Enzyme Kinetics of V. alginolyticus PepD (a) Michaelis-Menten plot for PepD catalyzed the hydrolysis of L-carnosine in 50 mM Tris-HCl, pH 6.8 at 37 ºC. (b) Lineweaver-Burk plot calculated from the respective Michaelis-Menten plot.

3.5 Site-directed mutagenesis analysis of Vibrio alginolyticus pepD

is analysis was

was notable that the conserved residue Asp119 of PepD was equal to the adjacent resi

In order to identify the putative active site of PepD, site-directed mutagenes

performed to investigate the essential amino acids. Recent studies reported that the active site residues of enzymes in M20 family were almost conserved.^{55, 58} Pep V, the first dinuclear dipeptidase with carnosine-hydrolyzing enzymatic activity protein in M20 family has been crystallized in 2002. PepD showed 20.9% identity and 34.3% similarity with PepV based on the sequence alignment employing. Sequence alignment between PepD and PepV also revealed the proposed active site. Surprisingly, the active site residues of PepV were almost conserved in PepD. These residues, including His80, Asp119, Glu150, Asp173, and His461 were expected for the metal binding, whereas Asp82 and Glu149 were expected for the catalysis (Fig. 8).

due Asp 120 of PepV that is the adjacent residue next to its metal binding residue Asp119.

It is common that aspartic acid residue exhibited the metal binding role extensively at the active site of the enzymes in M20 family. The PepV peptide group between the bridging Asp119 and the adjacent residue Asp120 exhibited a cis-conformation to affect the binding of the metal, whereas the Asp 119 from PepD might be considered to associate with two zinc ions simultaneously. The cis-conformation is thought to be necessary to force the important zinc bridging carboxylate into the correct geometry as described in 1.7. Therefore, the residue Asp119 of PepD may play a more important role on metal binding hence involve in the enzymatic activity. In the study Asp119 and another proposed catalytic residue Glu149 were initially investigated by site-directed mutagenesis.

Fig. 8 Multiple sequence alignment with PepD and PepV

are marked in black and gray,

p119,Glu150, Asp173, His461

The desired mutants were generated by QuickChange site-directed mutagenesis kit as described in 2.9 and the mutant plasm

Identical and conserved amino acids between the sequences

respectively. Dashed lines indicate the gaps introduced for better alignment.

▼ Proposed active site residues : Asp82 ,Glu149

＊ Proposed metal ion binding residues : His80, As

ids were transformed into E. coli. BL21(DE3)pLysS to express the mutant proteins. Following the same purification experimental procedure of V.

alginolyticus wild-type PepD, the mutant PepD proteins were carried out with 20 mM Tris-HCl pH 6.8 buffer containing 150 mM imidazole by Ni-NTA column chromtography.

The purified wild-type and mutant PepD proteins showed the same molecular weight about 55 kDa on SDS-PAGE (Fig. 9~10.).

-type and mutant proteins of D119.

ild type; Lane 2:PepD D119E mutant; Lane 19L mutant; Lane 5:PepD D119I mutant; Lane 19F mutant; Lane 8:PepD D119A; Lane 9:PepD Lane 11:PepD D119C mutant; Lane 12:PepD

Fig. 10 SDS-PAGE of E149.

Lane M:LMW protein marker; Lane 1:PepD wild type (WT); Lane 2:PepD E149A mutant;

Lane 3:PepD E149D mutant; Lane 4:PepD E149G mutant; Lane 5:PepD E149S mutant; Lane 6:PepD E149Q mutant; Lane 7:PepD E149H mutant; Lane 8:PepD E149R mutant

Fig. 9 SDS-PAGE (12%) of purified wild Lane M:LMW protein marker; Lane 1:PepD w 3:PepD D119M mutant; Lane 4:PepD D1 6:PepD D119 R mutant; Lane 7:PepD D1 D119S mutant; Lane 10:PepD D119T mutant;

D119P mutant; Lane 13:PepD D119N mutant

(12%) of purified wild-type and mutant proteins

M 1 2 3 4 5 6 7 M 8 9 10 11 12 13

30 97 66 45

M 1 2 3 4 5 6 7 8 97

66 45

In E. coli, PepD was indicated as a homodimer in native sate.²⁹ To observe the native form of PepD . alginolyticus, the wild-type and mutant PepD derivatives were analyzed with 7.5% Native-PAGE analysis (Fig. 11) A major band with molecular mass near 66 kDa of the protein marker was observed. Interestingly, several weak bands were also examined on the Native-PAGE of the wild-type and mutant PepD. Therefore, it was proposed that PepD in V. alginolyticus might exist in many forms in the native state. The major band with molecular weight about 66 kDa might be the monomer that PepD tended to form in its native state. The r form. To

estern blotting (Fig. 13). However, the calculated molecular weight from sedimentation coef

in V

minor weak band with molecular weight near 140 kDa might be the homodime

ensure the native form of PepD in V. alginolyticus, the Westeewrn blotting, that examining a clear band on the film was carried out using anti-PepD mAb (Fig. 12). Besides, Analytical Ultracentrifugation (AUC) was also used to confirm the result of Native-PAGE analysis and W

ficient (s) indicated that PepD preferred to form homodimer in its native state. The calculated molecular weight of denatured PepD protein was as a control comparing to the molecular weight of wild-type PepD protein (Fig. 14).

M 1 2 3 4 5 6

Fig. 11 Nativ-PAGE (10%) analysis of purified PepD wild-type and mutant proteins Lane M:HMW Native protein marker; Lane1: PepD wild-type(20 μL); Lane 2:PepD D119E mutant (20 μL); Lane 3:PepD E149D mutant (20 μL); Lane 4:PepD wild-type (40 μL); Lane

5:PepD D119E mutant (40 μL); Lane 6:PepD E149D mutant (40 μL) 232

140

M 1 2 3 4 5 6

0 100000 200000 300000 400000 500000 600000 700000 800000 0.000000

Fig. 12 Western blotting analysis of purified PepD wild-type and mutant proteins

Analytical ultracentrifugation determination of PepD protein

ecular 440

232 140

Lane M:HMW Native protein marker; Lane 1:PepD wild-type; Lane 2:PepD D119E mutant;

Lane 3:PepD E149D mutant; Lane 4:PepD wild-type; Lane 5:PepD D119E mutant; Lane 6:PepD E149D mutant

Fig. 13

The molecular weight of V. alginolyticus PepD is 53548.8 g/mol. The calculated mol weight from sedimentation coefficient (s) is about 96817.977 g/mol.

0 200000 400000 600000 800000 1000000 1200000 0.000000

0.000001 0.000002 0.000003 0.000004 0.000005 0.000006 0.000007

Data: Data1_B Model: Gauss

Chi^2/DoF = 4.1744E-15 R^2 = 0.99639

y0 0 ±0

xc 3783 4

5.70551 ±69.20846 w 190 9.24454 ±136.99134 A 0.1428 ±0.0009

PepD protein

The calculated molecular weight of denatured PepD protein from sedimentation coefficient (s) is about 37835.71 g/mol.

Fig. 14 Analytical ultracentrifugation determination of denatured

3.6 Enzyme kinetic of the mutant PepD

The enzyme activity of the mutant PepD was performed with hydrolyzing L-carnosine in e same experimental process of the wild-type PepD, as described in 2.6. Compared to the ild-type PepD, no apparent activity could be detected among almost all mutanted-PepD xcept the E149D mutant (Fig. 15).

WT E149D E149I E149G E149H E149A E149S E149W

Relative activi

Fig. 15 Enzymatic activities of wild-type and mutant PepD on L-carnosine. Purified wild-type and mutant PepD proteins were subjected to the activity assay on L-carnosine as a substrate. The wild-type activity was defined as 100%

Moreover, the enzyme kinetic of the mutants to determine the Vmax, Km and Kcat values, and compared to that of the wild-type PepD. The Vmax and Km values of the E149D mutant (2 μg, 0.186 μM) for L-carnosine calculated from the respective Lineweaver-Burk plot were 1.1 μM/min and 0.53 mM (Fig. 16).

hich catalyzed the hydrolysis of L-carnosine in 50 mM Tris-HCl, pH 6.8 at 37 ºC. (b) Lineweaver-Burk plot calculated from the respective Michaelis-Menten plot.

Fig. 16 Enzyme kinetics of wild-type and mutant PepD (a) Michaelis-Menten plot for wild-type and E149D mutant w

Therefore, the turnover number (kcat, kcat = Vmax/[E]T) of E149D for L-carnosine in 50 mM Tris-HCl, pH 6.8 at 37 ºC is 5.9 min^-1 and the catalytic efficiency (kcat/Km) is 0.186 mM^-1s^-1. Moreover, no activity at all could be detected for the D119E mutant (Table 6).

Table 6. Kinetic Parameters for the hydrolysis of L-carnosine at 37 ºC and pH 6.8 of wild-type and mutant V. alginolyticus PepD

PepD variant kcat (min^-1) Km (mM) kcat/ Km (mM^-1s^-1)

Wild-type 8.6 0.36 0.398

E149D 5.9 0.58 0.186

D11 ND ND ND

3.7 The secondary structure of Vibrio alginolyticus PepD

There is a common problem that the mutants created by using site-directed mutagenesis techniques might cause global conformational changes that inactivate the protein. Circular

在文檔中溶藻弧菌胺醯組胺酸雙胜肽酶之生化特性分析及其功能性胺基酸之研究 (頁 32-0)