# Patent

## Title
Fused NHase with improved specific activity and stability

## Abstract

The present invention provides a fused NHase with improved specific activity and stability, which relates to the field of genetic engineering. This invention provides a method of overexpressing a fused NHase in E. coli and producing a mutant NHase with improved stability and product tolerance. The invention provides a simple, efficient and safe method of making mutant NHase, and can produce a large amount of soluble NHases in a short period. The present invention makes a contribution to large-scale industrial production and further theoretical study of NHases.

## Background
CROSS-REFERENCES AND RELATED APPLICATIONS
This application claims the benefit of priority to Chinese Application No. 201510195795.0, entitled “A fused NHase with improved specific activity and stability”, filed Apr. 22, 2015, which is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the field of genetic engineering, and more particularly relates to a fused NHase with improved specific activity and stability.
Description of the Related Art
Nitrile hydratase (NHase; EC 4.2.1.84) is an enzyme catalyzing the hydration of a broad scope of nitriles to the corresponding amides. The NHase comprises a β-subunit, a α-subunit and a regulatory subunit and it is generally divided into the cobalt-type (Co-NHase) or the iron type (Fe-NHase) depending on the metal ion chelated with the active site.
NHase has been widely used in the industrial production of highly purified acrylamide and nicotinamide, since biotechnology synthesis has advantages of low-cost, low-energy consumption and less pollution compared to traditional chemical synthesis. However, most NHases with high activity are unstable during industrial application. For example, the NHases of Pseudomonas chlororaphils B23 and Rhodococcus sp. N-774 are unstable above 20° C., and the NHase of Rhodococcus rhodochrous J1 is merely stable between 10° C. and 30° C. In addition, it is necessary to maintain low reaction temperature to stabilize the NHases by refrigeration because of the exothermic reaction of nitrile-hydration, which usually causes enormous redundant energy cost. Furthermore, tolerance of NHase to high concentrations of the product is necessary in industrial manufacturing. Therefore, a more stable NHase with high activity and high tolerance is required for industrial manufacturing.
DETAILED DESCRIPTION
To solve the problems described above, the present invention provides a method of improving the specific activity, stability and tolerance of NHase. Usually, the subunits of NHase are separated, and they would be depolymerized at high temperatures which could result in enzyme inactivation. Therefore, the present invention fuses the β- and α-subunits with covalent bonds through molecular approaches, which eliminates the possibility of subunits depolymerization. The resulted fused NHase with improved stability is more suitable for using in the industrial production of acrylamide and the fusion strategy could be applicable for different NHases with separated subunits.
The present invention provides a mutant NHase with improved specific activity and stability. The β- and α-subunits of the mutant NHase are fused in the mutant NHase, and the regulatory subunit is either fused or coexpressed with the fused α- and β-subunits.
In one embodiment of the present invention, the nucleotide sequence of the mutant NHase from 5′ to 3′ is β-subunit gene (B gene), α-subunit gene (A gene), and regulatory subunit gene (P14K gene) fused together.
In one embodiment of the present invention, the mutant NHase is reconstructed from the parent NHase whose nucleotide sequence is SEQ ID NO: 1. And the amino acid sequences of the α-subunit, β-subunit and regulatory subunit of the mutant NHase are the same as those of the parent NHase from Pseudomonas putida NRRL-18668.
In one embodiment of the present invention, the amino acid sequences which encode the β-subunit, α-subunit and regulatory subunit are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The B gene and A gene are linked by a linker, and the nucleotide sequence of the linker is SEQ ID NO: 5.
In one embodiment of the present invention, the nucleotide sequence of the mutant NHase is SEQ ID NO.6 or SEQ ID NO.7.
The present invention also provides plasmids containing the amino acid sequences of the mutant NHase above and genetically engineered strains expressing the mutant NHase.
In one embodiment of the present invention, the genetically engineered strain is a recombinant E. coli BL21 (DE3).
The present invention also provides a method of constructing a genetically engineered strain expressing the mutant NHase.
In one embodiment of the present invention, the method of constructing the genetically engineered strain comprises cloning the nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7 to the expression plasmid of pET-28a to make a recombinant plasmid and transforming the recombinant plasmid into E. coli BL21(DE3).
The present invention also provides a method of producing NHases by the genetically engineered strain. The recombinant E. coli expressing the mutant NHase was cultivated in 2YT medium (tryptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L) at 37° C. When the optical density at 600 nm (OD600) of the culture reached 0.8, isopropyl-D-1-thiogalactoside (IPTG) and CoCl2.6H2O were added to the medium to induce the expression and maturization of NHase. The culture was subsequently incubated at 24° C. for 16 h.
The present invention also provides a method of improving the specific activity and stability of NHase, wherein the NHase is made by fusing the B and A gene together and coexpress the P14K gene, or by fusing the B, A and P14K gene together.
In one embodiment, the present invention provides a method to fuse the B, A and P14K gene from 5′ to 3′ in the order of “B gene, A gene, P14K gene”, and the B and A gene are connected by a linker whose nucleotide sequence is set forth in SEQ ID NO: 5.
The application of the mutant NHase, especially the application of the mutant NHase in acrylamide production is also under the scope of the present invention.
The mutant NHases obtained by the gene fusion strategy of the present invention exhibited significantly improved specific activity, thermostability and product tolerance than those of the wild type NHase.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1. SDS-PAGE of the wild type NHases and the fused NHases expressed by E. coli. Line 1, molecular weight marker; line 2, the wild type NHases; line 3, the mutant NHase-(BA); line 4, the mutant NHase-(BA)P14K; line 5, the mutant NHase-(BAP14K).
FIG. 2. Half-time in 50° C. of the wild type NHase and the fused NHases.
FIG. 3. Product tolerance of the wild type NHase and the fused NHases.
FIG. 4. The optimal pH of the wild type NHase and the fused NHases.



EXAMPLES
Materials and Methods:
2YT medium: 16 g·L−1 tryptone, 10 g·L−1 yeast extract, 5 g·L−1 NaCl.
The activity of NHase was detected by the method described as follows. The reaction mixture contained 500 μL 200 mM 3-cyanopyridine and 10 μl of the appropriate amount of the enzyme solution. The reaction was performed at 25° C. for 10 min and terminated with the addition of 500 μL of acetonitrile. Then the supernatant was collected by centrifugation and filtered though a 0.22 μm pore-size filter before measured by HPLC. One unit (U) of NHase activity is defined as the amount of enzyme that released 1 μmol nicotinamide per min under these assay conditions.
HPLC conditions: the mobile phase was water-acetonitrile buffer; detection wavelength was 215 nm; the column was C18 column.
Example 1 Construction of the Recombinant E. coli Expressing the Wild Type NHases-BAP14K
Construction of the recombinant E. coli expressing the wild type NHases-BAP14K was carried out by the following steps:
(1) Amplification of the parent NHase gene: Primers were designed according to the published sequence in NCBI to amplify the ABP14K gene encoding the parent NHase from P. Putida. The amino acid sequence of the β-subunit, α-subunit and regulatory subunit of the parent NHases were SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
(2) Construction of recombinant plasmid containing the ABP14K gene: the amplified DNA fragment of step 1 was digested with Nde I and Hind III, and then ligated into the Nde I and Hind III sites of pET-24a to create a recombinant plasmid containing the ABP14K gene. The recombinant plasmid was named pET-24a-ABP14K.
(3) Construction of recombinant plasmid containing the full-length BAP14K gene: pET-24a-ABP14K was used as a template. B gene was amplified by primer pairs B-up (SEQ ID NO: 8) and B-down(BA) (SEQ ID NO: 9), A gene was amplified by primer pairs A-up(BA) (SEQ ID NO: 10) and A-down(AP) (SEQ ID NO: 11), and the P14K gene was amplified by primer pairs P14K-up(AP) (SEQ ID NO: 12) and P-down (SEQ ID NO: 13). The same amount of B, A, P gene were used as templates and the full-length BAP14K gene was amplified by an overlap extension PCR protocol with primer pairs B-up (SEQ ID NO: 8) and P-down (SEQ ID NO: 13). The recombinant plasmid containing the BAP14K gene was named pET-24a-BAP14K, and the mutant NHase expressed by pET-24a-BAP14K was defined as the wild type NHase.
(4) Transformation of pET-24a-BAP14K into E. coli BL21 (DE3): The recombinant plasmid pET-24a-BAP14K was transformed into E. coli BL21 (DE3). The positive transformants expressing the wild type NHase were screened.
Primers used in the present invention were shown in Tab. 1.







TABLE 1







Primers













SEQ





ID



Primer
sequence (5′ to 3′)
NO















B-up
GGAATTC  AATG
8




GCATTCACGATACT




 



B-down
CATATCTATATCTCCTTTC
9



(BA)
ACGCTGGCTCCAGGTAGTC




 



A-up
TGAAAGGAGATATAGATAT
10



(BA)
GGGGCAATCACACACGC




 



A-down
CATATCTATATCTCCTTTT
11



(AP)
AATGAGATGGGGTGGGTT




 



P14K-up
TAAAAGGAGATATAGATAT
12



(AP)
GAAAGACGAACGGTTTC




 



P-down
CCG  TCAAGCCAT
13




TGCGGCAACGA




 



B-Nde I-
GGAATTC  AATGG
14



up
CATTCACGATAC




 



P-Hind
GCCC  TCAAGCCA
15



III-down
TTGCGGCAACGA




 



A-Hind
GCCC  TCAATGAG
16



III-down
ATGGGGTGGGTT




 



Linker1-
TACCTGGAGCCAGCGCCAGG
17



up
TGGGCAATCACACACGCAT




 



Linker1-
CGTGTGTGATTGCCCACCTG
18



down
GCGCTGGCTCCAGGTAGTC




 



Linker2-
CCCACCCCATCTCATCCAAA
19



up
TGGAGATATAGATATG




 



Linker2-
CATATCTATATCTCCATTTG
20



down
GATGAGATGGGGTGGG








Note: restriction sites were in italics and



bold; overlapping sequences were underlined.






Example 2. Construction of the Recombinant E. coli Expressing the NHase-(BA)P14K
The recombinant E. coli expressing the NHase-(BA)P14K was constructed by the following steps:
(1) The B and A gene were fused by linker 1 (SEQ ID NO: 5) by primer pairs Linker1-up (SEQ ID NO: 17) and Linker1-down (SEQ ID NO: 18) using pET-24a-BAP14K as a template. The resulted pET-24a-(BA)P14K was used as a template to amplify the (BA)P14K gene by primer pairs B-Nde I-up (SEQ ID NO: 14) and P-Hind III-down (SEQ ID NO: 15). The amplified (BA)P14K fragment was then digested with Nde I and Hind III, ligated into the Nde I and Hind III sites of pET-28a. The resulted recombinant plasmid pET-28a-(BA)P14K could express a fused NHase (nucleotide sequence shown in SEQ ID NO: 6), whose β- and α-subunits were fused together and the regulatory subunit was coexpressed. The NHase expressed by pET-28a-(BA)P14K was defined as NHase-(BA)P14K.
(2) The recombinant plasmid pET-28a-(BA)P14K was transformed into E. coli BL21 (DE3). Positive transformants expressing the NHase-(BA)P14K were screened.
Example 3. Construction of the Recombinant E. coli Expressing the NHase-(BAP14K)
The recombinant E. coli expressing the NHase-(BAP14K) was constructed by the following steps:
Primer pairs Linker2-up (SEQ ID NO: 19) and Linker2-down (SEQ ID NO: 20) were used to connect the A gene and P14K gene and pET-28a-(BA)P14K was the template. The resulted plasmid pET-28a-(BAP14K) contained a fused NHase gene whose B, A, and P14K gene fragments were fused together (nucleotide sequence shown in SEQ ID NO: 7). The NHase expressed by pET-28a-(BAP14K) was defined as NHase-(BAP14K).
The recombinant plasmid pET-28a-(BAP14K) was transformed into E. coli BL21 (DE3). The positive transformants expressing the NHase-(BAP14K) were screened.
Example 4. Expression and Characterization of the NHases
The E. coli recombinants obtained in example 1-3 were used to express the NHases.
The E. coli recombinants were firstly cultivated in 10 ml of liquid 2YT medium containing 50 μg/ml kanamycin at 37° C., then transferred to 500 ml of liquid 2YT medium with 1% inoculation. When OD600 of the culture reached 0.8, IPTG was added to a final concentration of 0.4 mM to induce NHase expression, and CoCl2.6H2O was added to a final concentration of 0.05 g/l to obtain mature NHase. The culture was subsequently incubated at 24° C. for 16 h and then the cells were harvested for SDS-PAGE.
Results indicated that the wild type NHase, NHase-(BA)P14K and NHase-(BAP14K) were successfully expressed, as shown in FIG. 1. The line 3 represented the mutant NHase-(BA) whose β-subunit and α-subunit were just fused in the absence of the regulatory subunit.
The characteristics of the subunits fused NHases:
Specific Activity
Determination of NHases was conducted by the following method. The E. coli recombinants were collected by centrifugation and resuspended with a 0.01M phosphate buffer (pH 7.5) twice before ultrasonic disruption. The enzyme in the supernatant was purified and then the enzyme activity was detected by HPLC.
Compared with 324.8 U/mg of the wild type NHase, the specific activity of NHase-(BA)P14K and NHase-(BAP14K) were 499.2 U/mg and 452.5 U/mg, which were increased by 53.7% and 39.3%, respectively. In addition, the specific activity of NHase-(BA) (FIG. 1, line 3) was 69.1 U/mg, indicating that the P14K was also necessary for cobalt incorporation in the fused NHase.
Furthermore, the kinetic parameters (Km, Vmax, kcat and kcat/Km) of NHase-(BA)P14K and NHase-(BAP14K) were compared with the wild-type NHase. Results showed that the kcat value of NHase-(BA)P14K (723.4 s−1) and NHase-(BAP14K) (676.5 s−1) were both approximately 2-fold of the wild-type NHase (335.1 s−1), indicating that the fused NHases exhibited faster catalyze rate. In addition, the kcat/Km value of NHase-(BA)P14K (11.8·103 s−1M−1) was about 1.5 fold of the wild-type NHase (8.1·103 s−1M−1), indicating higher catalytic efficiency of NHase-(BA)P14K.
Thermostability
The thermostability of the NHases was measured by the following steps. First, the eppendorf tube containing the enzyme solution was placed in a metal bath at 50° C. for a while before placed on ice. And then, the tube was placed at 25° C. in the metal bath and 200 mM 3-cyanopyridine (substrate) was added to it. Ten minutes later, acetonitrile was added to terminate the reaction.
As shown in FIG. 2, the half-life times of NHase-(BA)P14K and NHase-(BAP14K) were 26 min and 18 min, respectively, while that of the wild-type NHase was 9 min. Results suggested that the NHase-(BA)P14K and NHase-(BAP14K) exhibited higher thermostability than the wild-type NHase.
Product Tolerance of the NHases
The product tolerance of the NHases was measured by the following method. The reaction was conducted in 20 mM 3-cyanopyridine (substrate) with and without 0.5 M nicotinamide (product) for 10 min. The reduction of 3-cyanopyridine in each reaction was measured (FIG. 3), and the reduction ratio (the proportion of the reduced 3-cyanopyridine amount in the reaction with and without 0.5 M nicotinamide) was calculated.
Results showed that the consumption of substrate of NHase-(BA)P14K and NHase-(BAP14K) in product containing reaction systems were increased by 26% and 18%, respectively compared with the wild type NHase, and increased by 23% and 15% respectively in reaction systems without product. In addition, the reduction ratios of NHase-(BA)P14K (0.86) and NHase-(BAP14K) (0.83) were higher than that of the wild type (0.80), indicating that the fused NHases exhibited stronger product tolerance than that of the wild type.
The Optimum pH
The enzyme activities of the fused NHases were measured under different pH and compared with the wild type, and the activity under their respective optimum pH was defined as 1(100%). As shown in FIG. 4, the optimum pH of the three NHases were about 7.5.
These data showed that the specific activity, thermostability and product tolerance of NHase could be significantly increased by fusing the β subunit and the α subunit with the regulatory subunit fused or coexpressed at the same time.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.

## Claims

1. A mutant Nitrile hydratase (NHase) with improved specific activity and stability compared to its wild type NHase, wherein said mutant NHase comprises, from the N-terminus to the C-terminus, a β-subunit of SEQ ID NO: 2 and a linker encoded by a nucleotide sequence of SEQ ID NO: 5 fused to an α-subunit of SEQ ID NO: 3 in a single polypeptide, and a regulatory subunit of SEQ ID NO: 4.

2. The mutant NHase of claim 1, wherein said mutant NHase is encoded by a nucleotide sequence of SEQ ID NO: 6.

3. The mutant NHase of claim 1, wherein said mutant NHase is expressed by a plasmid.

4. The mutant NHase of claim 1, wherein said mutant NHase is expressed by a genetically engineered strain.

5. The mutant NHase of claim 4, the wherein said genetically engineered strain is Escherichia coli.

6. The mutant NHase of claim 4, the wherein said genetically engineered strain is constructed by the following steps:
a), cloning the nucleotide sequence of SEQ ID NO.6 to the expression vector of pET-28a to create a recombinant plasmid; and
b), transforming the recombinant plasmid into E. coli BL21.

7. A method of producing acrylamide, comprising using the mutant NHase of claim 1 to make acrylamide.