# Patent

## Title
Mutant with enhanced secretion of L-asparaginase and its application

## Abstract

The present invention provides a mutant with enhanced secretion of L-asparaginase and its application, which relates to the field of enzyme engineering. This invention successfully constructed a mutant with enhanced secretion of L-asparaginase through N-terminal deletion on amino acid sequence of L-asparaginase, and then expressed the mutant via a recombinant strain. Compared with the wild L-asparaginase, the secretion ability of L-asparaginase mutant in present invention significantly improves by 3.14 times compared to that of wild type. The recombinant strain that constructed in this prevention has an L-asparaginase yield of 407.6 U/mL and a production efficiency of 9.26 U/(mL/h), which reaches to the highest yield been reported.

## Background
CROSS-REFERENCES AND RELATED APPLICATIONS
This application claims the benefit of priority to Chinese Application No. 201510837174.8, entitled “A mutant with enhanced secretion of L-asparaginase and its application”, filed Nov. 25, 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 enzyme engineering, which relates to a mutant with enhanced secretion of L-asparaginase and its application.
Description of the Related Art
L-asparaginase (EC3.5.1.1) is an enzyme used effectively in the treatment of cancer. It catalyzes the hydrolysis of amino acid L-asparagine to aspartic acid and ammonia. L-asparaginase has been proved to have inhibitive activity on tumor cells, especially on acute leukemia and malignant lymphoma. Besides the cancer-combating properties and no inhibitory on bone marrow cells, L-asparaginase is employed as effective drugs in the treatment of acute lymphoblastic leukemia (ALL).
Two forms of L-asparaginases have been reported, namely L-asparaginase I and L-asparaginase II. The properties of these two enzymes, especially enzymes from Escherichia coli, Erwinia carotovora, and Erwinia chrysanthemi, have been studied intensively. Since it has been proved that only L-asparaginase II has cancer-combating properties, most research is about L-asparaginase II. L-asparaginase II produced by Escherichia coli and Erwinia chrysanthemi has already been developed into drugs for acute lymphoblastic leukemia treatment.
Acrylamide is formed through Maillard reaction which happens when sugars and asparagine are heated under high temperature. L-asparaginase can reduce the content of acrylamide in food.
L-asparaginase has been widely found in microbials, mammals and plants. Compared with the low content of L-asparaginase in animal serum, and the complex extraction process, there are advantages of L-asparaginase produced by means of microorganism fermentation, including easy cultivation and low costs. Current L-asparaginase is mainly produced by microorganisms including Escherichia coli, Erwinia carotovora, Erwinia chrysanthemi, etc. However L-asparaginase shows low yield in wild strains. In recent years, high efficient expression of L-asparaginase has become an important source for L-asparaginase production, which is realized by Escherichia coli expressed L-asparaginase gene.
However, the critical problem to be solved is to realize L-asparaginase expression in food safety strains and improve its secretion.
DETAILED DESCRIPTION
The goal of the present invention is to realize the L-asparaginase expression in Bacillus subtilis through a strong promoter p43 and an efficient secretory signal peptide. Furthermore, certain amino acids located in the N-terminal of L-asparaginase are truncated to promote the expression of L-asparaginase.
The first goal of the present invention is to provide a mutant with enhanced secretion of L-asparaginase, wherein the mutant comprises an amino acid sequence as shown in SEQ ID NO.1. The L-asparaginase mutant carries an exogenous signal peptide WapA that replaces the original signal peptide in nature L-asparaginase (NCBI No. NC-000964.3), and the N-terminal of the L-asparaginase mutant is deleted.
The mutant has an nucleotide sequence as shown in SEQ ID NO.2.
The second goal of the present invention is to provide a recombinant Bacillus subtilis which expresses the gene encoding L-asparaginase mutant via plasmid pP43NMK.
In one embodiment of the present invention, the L-asparaginase mutant gene carries nucleotides encoding exogenous signal peptide WapA.
In one embodiment of the present invention, the host of recombinant Bacillus subtilis is Bacillus subtilis WB600.
In one embodiment of the present invention, the L-asparaginase mutant gene comprises an amino acid sequence as shown in SEQ ID NO.2.
In one embodiment of the present invention, the L-asparaginase mutant gene is ligated to pP43NMK by restriction enzyme sites Kpn and Pst I and then transferred into Bacillus subtilis WB600.
The third goal of the present invention is to provide a method for producing L-asparaginase by the recombinant Bacillus subtilis. The seed culture is inoculated to the fermentation medium at an inoculation percent of 4% (v/v) and then cultivated at 37° C. to produce L-asparaginase.
In one embodiment of the present invention, the wherein method is carried out through maintaining the medium pH at 7.0 and maintaining dissolved oxygen at above 20%, and maintaining high density fermentation by adding sucrose and peptone during fermentation process.
The present invention provided a mutant with enhanced secretion of L-asparaginase through N-terminal deletion and a recombinant strain to express the mutant. Compared with the wild L-asparaginase, the secretion ability of L-asparaginase mutant in present invention significantly improves by 3.14 times. The recombinant in this invention has an L-asparaginase yield of 407.6 U/mL and a production efficiency of 9.26 U/(mL/h), which is the highest yield so far. The L-asparaginase production of the present invention is 4.5 times higher than the production of the recombinant Bacillus subtilis which expressed the L-asparaginase gene with wild type signal peptide (Cloning, expression, and characterization of L-asparaginase from a newly isolated Bacillus subtilis B11-06), and 8.71 times higher than that of recombinant E. coli (CN 201510102732.6).



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 illustrates the L-asparaginase activity of mutants.
FIG. 2 illustrates results of SDS-PAGE analysis of L-asparaginase; 1. Wild type; 2, WB43H; 3, WB43H-D30.
FIG. 3 illustrates time profiles for batch cultivation of recombinant D30 in 3 L fermenter; Open circles: the activity of asparaginase (U/mL); Closed circles: Cell growth; Open rhombus: Sucrose concentration (g/L).



DETAILED DESCRIPTION
Materials and Methods:
Medium:
LB medium: peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH was adjusted to 7.0.
Fermentation medium: soybean peptone 10 g/L, corn pulp 5 g/L, urea 1 g/L, sucrose 35 g/L, K2HPO4 2.3 g/L, KH2PO4 1.7 g/L, MgSO4 0.75 g/L, NaCl 5 g/L, pH was adjusted to 6.8-7.0.
Enzyme Assay:
L-asparaginase activity was determined by a spectrophotometric assay using asparagine as the substrate. One unit of enzyme was defined as the amount of enzyme that catalyzed the formation of 1 μmol NH3 per minute.
The standard assay comprised following steps: 1 mL 10 mmol/L K2HPO4—KH2PO4 (pH 7.5), 0.1 mL 189 mmol/L asparagine and 0.1 mL enzyme solution were mixed and incubated at 37° C. for 30 min. Then 0.5 mL 1.5 mmol/L TCA was added to terminate the reaction. The formation of NH3 was spectrophotometrically monitored at 436 nm by ShimadzuUV-1240. Enzyme activity was calculated according to the standard curve that obtained by (NH4)2SO4 detection.
EXAMPLE 1
Construction of Strain Possesses Strong Promoter with High Efficient Expression
Primers P1, P2 (shown in Table 1) were designed for amplifying gene L-ASP carrying signal peptide WapA. The plasmid pMA0911-wapA-SP-ansZ been constructed before was used as templates for amplifying gene L-ASP through polymerase chain reaction (PCR). The PCR cycle comprised: the first step at 98° C. for 3 minutes; 34 cycles of the second step at 98° C. for 30 seconds (denaturation), at 55° C. for 90 seconds (aling), and at 72° C. for 90 seconds (elongation). PCR was carried out using 50 μL of a reaction solution comprising 1 μL of each primer, 4 μL dNTP Mix, 10 μL 5×primeSTAR Buffer, 32.5 μL double distilled water, and 0.5 μL primeSTAR DNA polymerase.
The amplified DNA fragment was purified using gel extraction kit, DNA concentration of which was measured by agarose gel electrophoresis. The purified DNA fragment and plasmid pP43NMK was then cleaved at the restriction enzyme cleavage sites at both of its ends with Kpn I and Pst I. The resulting DNA fragment from L-ASP and pP43NMK was purified separately by gel extraction kit. DNA concentration were measured via agarose gel electrophoresis.
Subsequently, DNA fragments L-ASP were ligated to pP43NMK at a volume of 10 μL comprising 4 μL L-ASP, 1 μL carrier pP43NMK and 5 μL solution I. The ligation was carried out at 16° C. overnight then recombinant plasmid that designated as pP43H was obtained. The pP43H was then introduced into E. coli JM109. The resulting transformants were cultured on the LB agar containing ampicillin. Positive colonies were picked and plasmids of which were extracted. Transformants having a plasmid of interest were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. The recombinant plasmids WB43H were constructed through transforming pP43H into Bacillus subtilis commensurately according to above procedures.







TABLE 1







Primers









Name
Sequence
Number





P1
CGGGGTACCATTATAGGTAAGAGAGGAATGTA
SEQ ID



CACATGAAAAAAAGAAAGAGGCGAA
NO. 3


 


P2
AAAACTGCAGGGATCCTCTAGAGATTCGCCCT
SEQ ID 



AGG
NO. 4


 


P3
TCTGAAAAAAAGGATCTGCCAA
SEQ ID




NO. 5


 


P4
GTGGTGGTGGTGGTGGTGTT
SEQ ID




NO. 6









EXAMPLE 2
Construction of Strains with Truncated N-Terminal
Primers P3, P4 (shown in Table 1) were designed, plasmid pP43H was used as templates for amplifying truncated N-terminal DNA fragments through PCR. The PCR cycle comprised: the first step at 98° C. for 3 minutes; 34 cycles of the second step at 98° C. for 30 seconds (denaturation), at 55° C. for 90 seconds (aling), and at 72° C. for 90 seconds (elongation). PCR was carried out using 50 μL of a reaction solution comprising 1 μL of each primer, 4 μL dNTP Mix, 10 μL 5×primeSTAR Buffer, 32.5 μL double distilled water, and 0.5 μL prime STAR DNA polymerase.
The resulting PCR amplicons were purified and measured by agarose gel electrophoresis. The purified DNA fragments were dephosphorylated and ligated at 16° C. overnight adding with DNA ligase. The recombinant plasmid that designated as D30 was introduced into competent E. coli JM109. The resulting transformants were cultured on the LB agar containing ampicillin at 37° C. overnight. Positive colonies were picked and plasmids of which were extracted. Transformants having a plasmid of interest were identified based on the restriction enzyme cleavage patterns and via nucleotide sequence analysis. The recombinant mutant WB43H-D30 with 25 N-terminal deletion of gene L-ASP were constructed through transforming pP43H into Bacillus subtilis WB600 commensurately according to above procedures.
EXAMPLE 3
Verification of Highly Secreted Strain for L-Asparaginase Production
The recombinant WB43H, WB43H-D30 (constructed in example 1 and example 2) and pMA0911-wapA-SP-ansZ/B.subtilisWB600 (ZL201310716775.4) were inoculated in 10 mL LB medium containing kanamycin and shake cultured at 37° C. overnight. Culture broth were centrifuged at 4° C., 10000 r/min for 10 min, crude extracellular enzyme was obtained in supernatant, crude intracellular enzyme was obtained in supernatant of cell homogenates. Both extracellular and intracellular enzymes were used for enzyme assay.
The L-asparaginase activity was shown in FIG. 1. Compared with the wild type strain, the extracellular L-asparaginase activity in mutant WB43H was 39.52 U/mL, improved 1.4 times than that from the wild type; the extracellular L-asparaginase activity in mutant WB43H-D30 was 88.24 U/mL, improved 3.14 times than that from the wild type. The effect of improving L-asparaginase production through N-terminal deletion was further verified through SDS-PAGE analysis (FIG. 2).
EXAMPLE 4
Fed-Batch Fermentation for L-Asparaginase Production
The recombinant strain WB43H-D30 (constructed in example 2) was inoculated at an inoculation percent of 4% (v/v) and caltivated in 3-L fermentor to produce L-asparaginase. The initial medium has the same ingredient with the shake flask medium. The fermentation was carried out through maintaining pH at 7.0 by feeding acid and alkali, keeping the dissolved oxygen (DO) above 20% through controlling DO associating with agitation speed, and feeding sucrose and peptone for high density fermentation. As a result, the OD600 of culture broth was 153 after fermented for 40 h, L-asparaginase yield reached to 407.6 U/mL while productivity reached to 9.26 U/(mL/h) when fermented for 44 h (FIG. 3). This yield is 4.5 times higher than that of Bacillus subtilis carrying wild type signal peptide and 8.71 times higher than that of recombinant E. coli, which is the highest yield been reported so far. This high yield of L-asparaginase also indicated the suitability of Bacillus subtilis as a host for L-asparaginase production.
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. An L-asparaginase mutant comprising the amino acid sequence of SEQ ID NO.1.

2. An application of the L-asparaginase mutant of claim 1 in manufacturing medicine, comprising adding the L-asparaginase mutant into a predetermined formulation.