Inducing Expression and Reaction Characteristic of Nitrile Hydratase from Rhodococcus sp. SHZ-11

Chin. J . Chem. Eng.,15(4) 573-578

(2007)

Inducing Expression and Reaction Characteristic of Nitrile Hydratase from Rhodococcus sp. SHZ-l* WANG Chao(3,@)a,ZHANG Genlin(%&#)", XU Xiaolin($k/l\#)a and LI Chun(%&)bs** Department of Environmental and Biochemical Engineering, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, China School of Life Science and Technology, Beijing Institute of Technology, Beijing lOOO81, China

a

Abstract Inducing expression and the reaction characteristic of nitrile hydratase (NHase) from Rhodococcus sp. SHZ-I were investigated. The results showed that the expression of NHase was greatly enhanced with the cooperation of acrylonitrile and ammonium chloride as inducer in the medium and the specific activity of NHase was increased of 44%. Then the temperature, pH, concentration of acrylonitrile and acrylamide were evaluated, which affected the activity and reaction characteristic of NHase. It was found that the temperature and concentration of acrylamide were the most important factors for the catalyzation of NHase. The optimal catalysis temperature of NHase from Rhodococcus sp. SHZ-1 was 30'C, and the activation energy of the hydration of NHase was 90.2Wmol-' in the temperature range from 5°C to 30°C. K,,, of NHase was 0.095mol.L-' using acrylonitrile (AN) as substrate, and NHase activity was inhibited seriously when acrylonitrile concentration was up to 40g.L- , the substrate inhibition constant Kiis 0.283mol.L-'. Moreover, the NHase from Rhodococcus sp. SHZ-1 had very strong tolerance to acrylamide, in which the final concentration of acrylamide reached to 642g.L-' and the residual activity of NHase still maintained 8.6% of the initial enzyme activity. Keywords nitrile hydratase, biocatalysis, acrylamide, characteristic, Rhodococcus sp. SHZ- 1

1 INTRODUCTION Acrylamide (AM), a commercial chemical widely used to produce polymers in industrial processes of sewage treatment, petroleum recovery, papermaking and textile sizing. is manufactured from acrvlonitrile (AN) mainly by Lhemical processes[1,2]. Nitrile hydratase (NHase) produced by bacteria and fungi catalyzes the conversion of a large number of chemically diverse nitriles into amides and acids. The expression of NHase by microorganisms has already been used for the industrial production of AM and nicotinamide. NHase can also be used efficiently in environmental remediation by converting nitrile wastes into less toxic amides[3,4]. Great progresses have been achieved for years of effort in the screening and optimization of wild strains. Furthermore, some species of the wild strains have already been successfully applied in the industrial production of AM from AN[5,6]. Recently, it has been given more attention to increasing the activity and improving the stability of NHase in industrial biotransformations[7]. In this article, the strain Rhodococcus sp. SHZ-1 was used for AM production by means of AN hydration. This strain is particularly interested in AM production since the hydratase system exhibits high NHase activity and a poor amidase activity. Accordingly, very little amount of the AM was further transformed into the undesired acrylic acid in the course of the reaction. In our previous study, NHase from Rhodococcus sp. SHZ-1 was produced in the late stationary phase about 80-84h of the fermentation, which was harvested at the maximum NHase activity peak, as high as 942U.mg-' the specific activity (SA),

in small-scale reactors[8]. In order to improve the NHase activity from Rhodococcus sp. SHZ-1, the addition of inducer was considered, and the reaction characteristics of the NHase was also investigated.

2 MATERIALS AND METHODS 2.1 Strain and culture conditions Rhodococcus sp. S H Z was screened under the saline and alkaline environment from Xinjiang. And based on the Rhodococcus sp. SHZ, the Rhodococcus sp. SHZ-1 strain was rebuilt in the extremely directional cultivation. These strains were conserved by the Lab. of Green Chemical Technology in Shihezi University. The colony of Rhodococcus sp. SHZ-1 was orange and had glossy surface. 50ml nutrient broth culture was used to inoculate 250ml damper flasks. The composition of the medium was as follows:, 2 0 g ~ ' of Glucose, 5g.L-I of yeast extract, 2g.L- NaCli 0.5g.L-' KH2Po4, 0.5~.L-' &HP04*3H20, 0.2g.LMgS04.7H20, 18g.L- of agar. The pH of medium was adjusted to 7.2. The medium was sterilized by autoclaving at 121 "C for 15min[9,10]. Inoculation preparation of Rhodococcus sp. SHZ-1 was performed in medium without adding agar. After culture at 30"C, 200r.min-' for 48h, 10% of inoculation was transferred to 1000ml fermentation flasks. All the chemicals were commercially available reagents of analytical grade.

2.2 Determination of biomass The biomass was rapidly estimated by optical density measurement at 600nm wavelength. Dry cell mass were determined by drying the wet cells until a

Received 2006-03-26, accepted 2007-05-28.

* Supported by the National Natural Science Foundation of China (No.20466002), the Program for New Century Excellent Talents in University (NCET-04-089) and the Key Research Projects in the Uygur Autonomous Region of Xinjiang (No.200332109).

** To whom correspondence should be addressed. E-mail: [email protected]

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constant dry mass (at 80°C)[111. From the calibration curve, one OD6oounit of Rhodococcus sp. SHZ-1 suspension corresponds to 0. 188mgrnl-'. Zero absorbance was set using distilled water. All the operations were replicated at least twice and average values are reported in this work.

2.3 Induction of NHase from Rhodococcus sp. SHZ-1 Induction of NHase was performed by added certain amount of inducers to culture flasks during the scheduled period of fermentation. The concentration of inducers tested were: AN (25, 50, 75mmyl.L-'), sodium acetate (15, 30, 50, 75mmol.L - ) and NH4Cl( 15, 30, 50, 75mmol.L - I ) solutions. These chemicals have been successfully used as inducers in other studies[12-15]. The flask cultures were started with 10% of inoculation biomass concentration. The SA of NHase was measured every 4h during the cultivation. The control was a treatment without addition of inducer.

2.4 Reaction characteristic of NHase from Rhodococcus SP.SHZ-1 The activity of NHase was investigated using 4.75 mg of cells collected from the culture broth,at the stationary phase and treated with 2OOmmol-L- AN, which indicated negligible substrate inhibition. The effect of pH on reaction rate was investigated in the range of pH 4.0-9.6, with 25mmol.L-' citrate buffer (PH 4.0-5.6), 25mmol.~-'~a21-1~04 /N&P04 (PH 6.0-7.6) and 25mmol.L;' K2HP04 /KH2P04 buffer (pH 8.0-9.2). 200r.min- and 30°C were selected to minimize deactivation caused by shear stress and temperature. The effect of temperature on the NHase activity was studied in the reaction temperature varied from 5 to 50°C. The effect of substrate concentration on the NHase activity was studied in AN solution, at concentration from 10 to lOOg.L-', which was prepared with 25mmol-L-' K2HP04 /KH2PO4 buffer (pH 7.2). Inhibition studies on NHase activity were carried out by pre-incubating resting cells with initial AM concentrations between 100-500g.L-'. At predetermined time intervals (20min) a sample was withdrawn and washed three times with 25mmol*L-' KZHPo4/KH2P04 buffer (pH 7.2), then NHase activity was analyzed[l l,16,171. 2.5 Resting cell preparation Cells were harvested at optimal fermentation times. The broth was centrifuged, at 12000r.min-' for lOmin at room temperature, and separated cells were washed three times using 18ml of 25mmol.L-' K2HPO4 / KH2PO4 buffer with pH 7.2. The cell paste was then suspended in the same buffer and kept in a refrigerator (-4°C) for the rest reaction.

2.6 NHase activity assay One unit (U) of NHase activity was defined as the amount of resting cells that catalyzed the formation of lpmol of AM per minute under the adopted August, 2007

conditions. Specific activity (SA) was denoted as U*rng-'[18,19]. NHase activity of resting cells was measured at 30°C for 5min by direct assay. The reaction mixture was composed of lml of 2OOmmol.L-' AN as subftrate, lml resting cells and 18ml of 25mmol.L- K*HPO&HzP04 buffer with pH 7.2. After incuption at 30°C with stirred continuously at 200r.min- for 5min the reaction was halted by adding lml of 4mol.L-'HCl. Then the reaction mixture was centrifuged at 12000rmin-' for lOmin, and the supernatant was used for the determination of product and residual substrate. The concentration of AN and AM in the reaction system were measured by gas chromatography (GC-2010, SHIMADZU), with a 2mX4mm column (5% Advance DS). The temperature of column, injector and flame ionization detector were 170°C, 200"C, 200"C, respectively, and the flow rate of nitrogen gas was 20ml*min-'.

3 RESULTS AND DISCUSSION 3.1 Inducing expression of NHase from Rhodococcus SP. SHZ-1 It was known that biomass and NHase activity were related to different inducers and their concentrations. The effect of different inducers on enzyme expression was shown in Fig.1. The highest NHase activity in the cells of Rhodococcus sp. SHZ-1 was obtained by i n p i n g of NH&1 and AN when N h C l (75mmol.L- ) was added into medium at the beginning of fermentation and AN (50mmol.L-') was added after 48h of cultivation. SA and total enzyme activity of treatment with inducer were increased obviously to 1358U*rng-' (Fig.2) and 8210Um-', respectively. According to what had been reported, SA from this strain was the highest one[8,20]. Moreover, cells could be harvested at the maximum enzyme activity (60h), and it was saved about 20-24h compared with the control (Fig.2).

-

I.Ann

K

$ .-

c

1200

.-

+d

1 5

8

'Oo0

800 600 control

NH,CI CH,COONa AN

NH,CI+AN

inducer

Figure 1 The effect of different inducers on enzyme production (Horizontal bar indicates the type of inducers: 75mmolC' W C l , 30mmol.L-' CH3COONa,5Ornrnol.L-' AN, 75mmol.L-' NH&I and 50 mmol. L-' AN. Errors bar represents SA of Rhodococcus sp. SHZ- 1. The control was a treatment without addition of inducer)

Inducing Expression and Reaction Characteristic of Nitrile Hydratase from Rhodococcus sp. SHZ-1

24 32 40 48 56 64 72 fermentation time, h

Figure 2 Biomass and SA curve of Rhodococcus sp. SHZ-1 in shake flasks (100Oml)induced by 75mmol*L-' NH4CI and SOmmol.L-' AN o specific NHase activity; B biomass concentration

Effects on NHase activity and biomass accumulation were also observed by adding NH4Cl or AN separately. After 72h incubation, biomass reached 6.22mg.d' with NHfl (75mmol-L-') and 6.35mg.d' with AN (50mmql.L- ), respectively, and SA of YHase was 1024U.mg- with NH4Cl, and 1136U-mg- with AN, higher than that of the control (942U*mg-') (Fig.1). However, it took longer time to reach the maximum enzyme activity comparing with addition of NH4Cl and AN. Otherwise, sodium acetate had not any effect on the NHase activity under chosen concentrations. It was clear (Figs.1 and 2) that addition of AN and NH4Cl into the medium were necessary for the high expression of NHase, where the SA was increased of 44% compared with the control. According to reported, AN has modestly effective on inducing aneuploidy chromosome loss, and has a structural feature that the cyano group CN is adjacent to a doubly bonded carbon atom ==C-CN[13,21,22]. Amide might devote into the puckering activity site and keeping stabilization of the NHase. Therefore, the better effect of NHase activity from Rhodococcus sp. SHZ-1 could be mainly attributed to cooperation of AN and NH4Cl in this research.

575

4.0 4.8 5.6 6.4 7.2 8.0 8.8 9.6 PH

Figure 3 The effect of pH on the activity of NHase in resting cells (Runs at 3O"C, 4.75mg dry cells and 2OOmmol~L~' AN in 25mmol.L-' citrate, Na2HP04/NaH2P04and K2HP0&H2P04 buffer)

the industrial scale, when costly would be the equipments need to keep pH within a narrow range.

3.3 Effect of temperature on enzyme activity The kinetic parameters and the activation energy of AN conversion to AM were determined. Fig.4 displayed the effect of temperature on the activity of NHase from Rhodococcus sp. SHZ-1 resting cell. The specific reaction rate, v, was evaluated from plots of product concentration against temperature. Here the values varied with temperature according to the known Arrhenius equation[22-251:

(

v=Aexp -:TI where A is the pre-exponential factor, E is the activation energy, R is the gas constant, and T is the absolute temperature. Fig.5 indicated the Arrhenius plot of AN hydratation catalyzed by NHase from Rhodococcus sp. SHZ-1 resting cell. apparent activation energy of roughly 90.2k.l-mol- was evaluated in the temperature range from 5 to 30°C.

+I

50 -

ei

0-

- :e

40-

h ' M

3.2 Effect of pH on enzyme activity A previous study showed that the change of NHase activity while the pH ranging from 6.0 to 8.0 was almost negligible[23]. The present investigation aimed to verify the effect of pH on the activity of NHase from Rhodococcus sp. SHZ-1. The effect of pH on reaction rate was showed in Fig.3. The results indicated that the highest specific reaction rate was obtained at pH 7.2, and the rate was slightly affected in the ranging of pH from 6.4 to 7.6. Beyond this range, the NHase activity decreased drastically, with NHase activity near to nil at pH=4.0. It was concluded that this strain could be used to catalyze hydration reaction in a relatively broad pH ranges between 6.4 and 7.6. The bioconversion of AN with NHase from Rhodococcus sp. SHZ-1 could allow certain pH fluctuation without loss of performance during an industrial process. This was rather important since exact pH control was difficult and more expensive in

.s2 30 0

2 E 20-

0I

0

10

I

I

I

20 30 40 temperature, 'C

I

50

Figure 4 The effect of temperature on the specific reaction rate of NHase in resting cells (Running with 4.725 mg dry cells and 20Ommol.L-' AN in 25rnrnol K' K2HP0&H2P04buffer, pH7.2)

According to the curve of residual activity under different temperatures (between 20 "C and 50 "C ), catalyzing reaction by NHase in resting cells exhibited a first-order deactivation mechanism[26] described by r, = v exp (-kdt) (2) The first-order deactivation constant of NHase, Chin. J. Ch. E. 15(4) 573 (2007)

Chin. J. Ch. E. (Vol. 15, No.4)

576 0

-1

d

-

-2

v

C

-3

-4 3.30

3.35

3.40

3.45

3.50

3.55

3.60

T 'XIO',K'

Figure 5 Arrhenius plot of AN hydratation catalyzed by NHase from Rhodococcus sp. SHZ-1 resting cells (Insert shows the dependence of inactivation constant on temperature)

kd , which depended on the relative activity (I*) and the temperature, could be evaluated according to the linearized version of the above equation. The insert in Fig.5 showed that kd values changed with temperature, too. As a result, NHase appears to be a thermolabile enzyme which lose its activity quite rapidly up to 40°C (the half-lives were 2.3h at 40°C and 0.98h at 5OoC, respectively). Better stability was obtained at 25'C and 35°C (half-lives were 6.31h and 5.45h, respectively). Take the deactivation constant, k d and reaction rate into consideration, 30°C was chosen as the optimal temperature for the activity and stability of NHase.

3.4 Effect of AN concentration on the enzyme activity The effect of substrate concentration on specific reaction rate of NHase was shown in Fig.6. An apparent maximum of specific reaction rate was 27.99polmin-'-mg-' with the concentration of AN 3Og.L-'. Whereas, the specific reaction rate dropped gradually when the concentration of AN was over 30

-0

+

0

20 40 60 80 100 substrate concentration, g. L-'

Figure 6 The effect of substrate concentrations on specific reaction rate August, 2007

4Og*L-', which reduced to 12.3~ol.min-'~mg-'at the substrate level of 1OOg-L- . It indicated that NHase activity was significant inhibited by high concentration of AN. Similar observations on nitrile utilization had been reported by Nagasawa and his co-workers using Rhodococcus rhodochrous J 1, in which hydratase activity was completely inhibite? by increasing the nitrile concentration up to 70g.L- [3]. In contrast, Rhodococcus sp. SHZ-1 still had 23% NHase activity residue under the substrate concentration of lOOgL-', which showed NHase from Rhodococcus sp. S H Z - 1 had very strong AN tolerance. According to the results of experiments and the characteristic of substrate at high concentration, it was speculated that the high concentration of AN might combine with enzyme and further form the ternary complex, so the catalysis rate of enzyme was reduced, and this inhibiting reaction belongs to the type of uncompetition inhibition[27,28]. This inhibition results in an apparent decrease in both ,v and K,. The equilibrium model of uncompetitive is given by 'ES k+2 , P + E E+S' k.,

where E is the enzyme, S is the substrate, ES is the enzyme-inhibitor complex, P is the product, ES2 is the enzyme-inhibitor-substratecomplex. The traditional equilibrium constant for this reversible reaction is given by

Inducing Expression and Reaction Characteristic of Nitrile Hydratase from Rhodococcus sp. SHZ-1

k K . =-3

'

(3)

k+3

When the inhibitor model is the type of uncompetitive, an equation for the inhibition scheme is

577

centration of AM, the more significant inhibition on NHase activity was observed. This inhibition was quite severity especially at concentration of 500g.~-' AM as indicated in Fig.S(b). The curve showed an apparent maximum of specific reaction rate of 20.6pmolmin-'.mg-' at 1OOg.L-'AM, but dropped to 1.82pmolmin-'.mg-' at 5OOg.L-' AM after 100min. It was found that Rhodococcus sp. SHZ-1 strain still retained 8.6% enzyme activity comparing with that of Rhodococcus sp. SHZ.

By rearrangement Eq.(4) yields

+)

When [ S ] 6 K i ,the Eq.(5) reduces to 1=

-(

1

1+

20

Vmax

40 60 80 hold time, min

(a) Rhodococcus sp. SHZ

where [Slis the concentration of substrate, vmaxis the maximum reaction rate, and v is the specific reaction rate, K, is the Michaelis constant. Under these conditions, The Michaelis constant of NHase from Rhodococcus sp. SHZ-1 was evaluated using the Lineweaver-Burk figure. According to the figure, the K , of NHase was 0.095mol.L-' using AN as substrate. When [ S ]3 K,, the Eq.(5) reduces to

I

20 \

,

Using the Eq.(7), the plot of substrate concentrations versus reciprocal of specific reaction rate was showed in Fig.7, the substrate inhibition constant Ki was 0.283mol.L-'.

100

I

40

I

60 80 hold time, min

I

100

(b) R ~ O ~ O C O CSP. C WSHZ-1 Figure 8 The effect of AM concentration on the NHase activity of Rhodococcus sp. SHZ and Rhodococcus sp. SHZ-1 AM concentration, g.L-': 100; 0 200; A 300; m400; 0 500

Figure 7 The plot of substrate concentrations versus reciprocal of specific reaction rate

Further experiments showed that the tolerance of Rhodococcus sp. SHZ-1 strain to AM had been improved comparing with the Rhodococcus sp. SHZ. The average specific reaction rate was enhanced by 37.5% faster than that of Rhodococcus sp. SHZ. Also, the hydration rate of AN by free cells from Rhodococcus sp. SHZ-1 was 26.6% faster than the Rhodococcus sp. SHZ, and the hydration concentration of AM reached to 642g.L-I. It was convincingly shown that the Rhodococcus sp. SHZ-1 had high expression of NHase activity, as well as strong tolerance of AM. It means that Rhodococcus sp. SHZ-1 has potential application in the synthesis of AM.

3.5 Effect of AM concentration on the enzyme activity Our previous studies indicated that Rhodococcus sp. SHZ resting cells could not tolerant high AM concentration[9]. Thereby, the present study was to ascertain whether Rhodococcus sp. SHZ-1 has high AM tolerance or not. As shown in Fig.8, AM had a negligible inhibition for the NHase (both Rhodococcus sp. SHZ and Rhodococcus sp. SHZ-1 strains) activity at concentrations as low as 2OOg.L-I. However, the higher con-

CONCLUSIONS Rhodococcus sp. SHZ-1 was a good strain to produce NHase. It was necessary to add AN and N h C l into the medium for inducing the expression of NHase, and the SA was increased of 44% comparing with control, under the cooperation with two inducers (75mmol.L-' N h C 1 and 5Ommol.L-' AN). Experimental results also verified that nitriles were effective compounds in terms of their induction expression of NHase. As the reaction characteristic of Rhodococcus sp.

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0.08

0.05

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0.03 0

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80 100 substrate concentration, g.L I 20

40

60

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SHZ-1 were concerned, the temperature and concentration of AM were found to be the most important factors for the catalyzation of NHase. The optimal catalysic temperature of NHase from Rhodococcus sp. SHZ-1 was 30°C, and the higher temperature, the quicker devitalizing rate. The activatipn energy of the hydration of NHase was 90.2kJ.mol- in the temperature range from 5’C to 30°C. K,,, of NHase was 0.095mol.L-’ using AN as substrate, and NHase activity was inhibited seriously when the concentration of AN was up to 4Og.L-’ the substrate inhibition constant K , is 0.283mol-L-’.’ Moreover, the NHase from Rhodococcus sp. SHZ-1 could tolerate the AM under the concentration of 5OOg-L-I. The final, hydration concentration of AM reached to 642g.L- , and the residual activity of NHase was about 8.6% of the initial enzyme activity after 100min. It was fully confirmed that the Rhodococcus sp. SHZ-1 strain had high expression of NHase activity, as well as strong tolerance of AM.

NOMENCLATURE A E Ki

pre-exponential factor, mm01.L- lmin-l activation energy, Jmol-l inhibition constant, mo1.L-l K, Michaelis constant, mo1.L-l kd deactivation constants, h-‘ R gas constant, J.K-’.mol-’ relative enzyme activity, % concentration of substrate, mo1.L T absolute temperature, K t time, h v specific reaction rate, pmol.min-l.mg-’ vmax maximum reaction rate, pnolmin-lmg-’

?

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