Screening method of bacillus thuringiensis for producing (+) gamma-lactamase

Abstract

The invention belongs to the technical field of microbial screening, and discloses a screening method of bacillus thuringiensis for producing (+) gamma-lactamase. The method comprises the following steps: taking (+ / -) gamma-lactam as a unique carbon source, carrying out primary screening by using a plate screening method, carrying out secondary screening by using a high performance liquid chromatography technology, successfully screening a strain for producing (+) gamma-lactam from a deep sea mud sample, and identifying the strain as bacillus thuringiensis. Reference is provided for development and utilization of marine resources, and a new way is provided for a strain for producing (+) gamma-lactamase.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganism screening, and relates to a method for screening Bacillus thuringiensis producing (+)-γ-lactamase. Background Art

[0002] γ-Lactamase (EC 3.5.1.4) is defined according to its enantioselectivity for (±)-γ-lactam, and is an enzyme that selectively cleaves the amide bond of amide compounds. Abacavir and carbovir are effective drugs for treating diseases caused by human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Currently, these drugs are synthesized using optically pure γ-lactam as a synthon. γ-Lactam usually exists in the form of a racemate. Since different enantiomers have different or even opposite pharmacological effects, it is usually not directly used in drug production. Therefore, it is necessary to resolve the racemate (±)-γ-lactam. Biocatalytic resolution has the characteristics of mild reaction conditions, high optical purity of products, and little environmental pollution, and is widely used in the resolution of (±)-γ-lactam.

[0003] In the research on the biocatalytic resolution of (±)-γ-lactam, strains with high enantioselective γ-lactamase are usually screened from the environment. However, there are few strain resources of (+)-γ-lactamase at present, especially the strain resources of (+)-γ-lactamase from marine sources are rarely reported. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a method for screening Bacillus thuringiensis producing (+)-γ-lactamase. Using (±)-γ-lactam as the sole carbon source, the primary screening is carried out by the plate screening method, and the secondary screening is carried out by high performance liquid chromatography technology. A strain producing (+)-γ-lactam is successfully screened from deep-sea sediment samples and identified as Bacillus thuringiensis. This invention provides a reference for the development and utilization of marine resources and a new way for strains producing (+)-γ-lactamase.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] A method for screening Bacillus thuringiensis producing (+)-γ-lactamase, using (±)-γ-lactam as the sole carbon source, the primary screening is carried out by the plate screening method, and the secondary screening is carried out by high performance liquid chromatography technology, and a strain producing (+)-γ-lactam is screened from deep-sea sediment samples.

[0007] A method for screening Bacillus thuringiensis producing (+)-γ-lactamase, the specific steps are as follows:

[0008] Step 1, primary screening of strains

[0009] Taking sea mud as a sample, 5.0 g of the sample was added to 45 mL of sterile physiological saline with a mass fraction of 0.85%, and after mixing, it was inoculated into an enrichment medium for enrichment culture at an inoculation amount of 5% by volume, and cultured at 28 °C and 150 r / min for 3 d; the enriched bacterial liquid was serially diluted, and 100 μL was taken from each of the bacterial liquids with dilution factors of 10 -5 , 10 -6 , 10 -7 and spread on a solid screening medium, cultured at 28 °C for 3 d, and single colonies were picked for streak purification; the primary screening strains were obtained;

[0010] Step two, rescreening of the strains

[0011] The primary screening strains were inoculated into a seed medium and cultured at 28 °C and 150 r / min for 1 d; inoculated into the initial fermentation medium at an inoculation amount of 5% by volume, cultured at 28 °C and 150 r / min for 1 d, and then the enzyme activity was measured by chiral HPLC, and then the strains with high enzyme activity were selected and preserved in 50 vt% glycerol at 1 / 1 (v / v);

[0012] Step three, colony characteristics

[0013] Step four, molecular biological identification of the strains; after extraction of genomic DNA, PCR amplification, sequencing, and sequencing alignment, the species of the strains were determined;

[0014] Step five, preservation of the strains.

[0015] Furthermore, in step one, the formula of the enrichment medium is: yeast extract 5 g / L, (±)γ-lactam 10 g / L, NH 4 Cl 10 g / L, NaH 2 PO 4 1 g / L, Na 2 HPO 4 1 g / L, MgSO 4 1 g / L, agar powder 20 g / L, pH 7.0

[0016] Furthermore, in step one, the formula of the solid screening medium is: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, (±)γ-lactam 3 g / L, agar powder 20 g / L, pH 7.0

[0017] Furthermore, in step two, the formula of the seed medium is: yeast extract 5 g / L, glucose 2 g / L, KH 2 PO 4 7 g / L, Na 2 HPO 4 2 g / L, MgSO4 0.4 g / L, FeSO 4 0.008 g / L, CaCl 2 0.01 g / L, N-acetyl-L-phenylalanine 3 g / L, pH 7.0

[0018] Furthermore, in step two, the enzyme activity in the high-enzyme-activity strain is above 655 U / mL.

[0019] Furthermore, step three is specifically as follows: Streak the high-enzyme-activity strain screened in step two on a solid LB (Luria-Bertani) medium, culture at 28 °C for 1 - 2 d, observe the colony morphological characteristics of the strain, pick a single colony, perform Gram staining, and observe the cell morphology of the bacteria.

[0020] Furthermore, in step four, the genomic DNA is extracted using a bacterial genomic DNA extraction kit.

[0021] Furthermore, in step four, after PCR amplification and sequencing, specifically, the amplified product is sent to Meiji Biotechnology Co., Ltd. for sequencing, and the primer sequences are: 27F: 5’-AGTTTGATCMTGGCTCAG-3’, 1492R: 5’-GGTTACCTTGTTACGACTT-3’.

[0022] Furthermore, in step four, the strain genus is determined by sequencing alignment and then through gene sequence homology analysis and the construction of a phylogenetic tree. Use the BLAST sequencing alignment results, download the sequences of highly homologous strains, and construct a phylogenetic tree using MAGE 11 software.

[0023] Furthermore, step five is specifically as follows: Activate the preserved strain to the logarithmic phase, mix the bacterial liquid with 30% (v / v) glycerol at a ratio of 1:1, and store at -20 °C.

[0024] The deep-sea mud sample is from the mud in the Bohai Bay of Dalian, Liaoning Province.

[0025] The above screening method screened a strain producing (+)-γ-lactamase from deep-sea mud, which was identified as Bacillus thuringiensis. It is a strain producing (+)-γ-lactamase from a marine source.

[0026] The screened Bacillus thuringiensis producing (+)-γ-lactamase is a strain resource producing (+)-γ-lactamase and can be applied in the biomedical field for the biocatalytic resolution of (±)-γ-lactam.

[0027] The screened Bacillus thuringiensis producing (+)-γ-lactamase is used for fermentative production of (+)-γ-lactamase, and (+)-γ-lactamase with relatively high enzyme activity can be obtained.

[0028] The beneficial effects of the present invention compared with the prior art are as follows:

[0029] The invention uses (±)-γ-lactam as the sole carbon source, and initially screens by the plate screening method and re-screens by high performance liquid chromatography technology, and successfully screens a strain producing (+)-γ-lactam from deep-sea mud samples, which is identified as Bacillus thuringiensis. The invention provides a reference for the development and utilization of marine resources and a new way for strains producing (+)-γ-lactamase. Description of the Drawings

[0030] Figure 1 It is the colony morphology diagram of strain LHN-5-3B in LB solid medium.

[0031] Figure 2 It is the diagram observed under an oil immersion microscope (10×100 magnification) after Gram staining. Detailed Embodiments

[0032] The present invention is described in detail below by specific embodiments, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained from commercial channels.

[0033] Example 1

[0034] A screening method for Bacillus thuringiensis producing (+)-γ-lactamase;

[0035] Step 1, initial screening of strains

[0036] Using sea mud as a sample, each time 5.0 g of the sample is added to 45 mL of sterile physiological saline with a mass fraction of 0.85%, and after mixing, it is inoculated into an enrichment medium at an inoculation amount of 5% by volume, and cultured at 28°C and 150 r / min for 3 d. The enriched bacterial liquid is serially diluted, and 100 μL is taken from the bacterial liquid at dilution factors of 10 -5 10 -6 10 -7 and spread on a solid screening medium, cultured at 28°C for 3 d, and single colonies are picked for streak purification.

[0037] Step 2, re-screening of strains

[0038] The initially screened strains were inoculated into the seed medium and cultured at 28°C and 150 r / min for 1 day. Then they were inoculated into the initial fermentation medium at an inoculation amount of 5% (v / v) and cultured at 28°C and 150 r / min for 1 day. Subsequently, chiral HPLC was used to measure the enzyme activity, and then the strains with high enzyme activity were selected and preserved in 50% glycerol at 1 / 1 (v / v).

[0039] Step 3, Colony characteristics

[0040] The screened strains were streaked on solid LB (Luria - Bertani) medium and cultured at 28°C for 1 - 2 days. The colony morphological characteristics of the strains were observed, and single colonies were picked for Gram staining to observe the cell morphology of the bacteria.

[0041] Step 4, Molecular biological identification of the strains

[0042] A. 16sRDNA sequencing: Sent to Sangon Biotech Co., Ltd. for sequencing. The primer sequences were: 27F: 5’ - AGTTTGATCMTGGCTCAG - 3, 1492: 5’ - GGTTACCTTGTTACGACTT - 3’.

[0043] The sequencing results were shown in GenBank: PV083904.1

[0044] B. Gene sequence homology analysis and construction of phylogenetic tree

[0045] The sequencing results were compared using BLAST. The sequences of strains with high homology were downloaded and used to construct a phylogenetic tree by NCBI to determine that the strain was Bacillus thuringiensis.

[0046] Step 5, Strain preservation

[0047] The bacteria were activated to the logarithmic phase and preserved in 30% glycerol at - 20°C.

[0048] The above - described embodiments are only the preferred embodiments of the present invention, rather than all the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious changes made without departing from the principle and spirit of the present invention should be considered to be included within the protection scope of the claims of the present invention.

Claims

1. A method for screening Bacillus thuringiensis producing (+) γ-lactamase, characterized in that: Using (±)γ-lactam as the sole carbon source, the plate screening method was used for primary screening and the high performance liquid chromatography technology was used for secondary screening to screen out (+)γ-lactam-producing strains from deep-sea mud samples.

2. The method for screening Bacillus thuringiensis producing (+) γ-lactamase according to claim 1, characterized in that: The specific steps are as follows: Step 1: Initial screening of strains Using Bohai Sea mud as a sample, 5.0 g of the sample was added to 45 mL of 0.85% sterile saline, mixed and inoculated into an enrichment medium at a volume ratio of 5% for enrichment culture, and cultured at 28°C and 150 r / min for 3 days; the enrichment culture solution was gradiently diluted from 10 -5 , 10 -6 , 10 -7 Take 100 μL of each diluted bacterial solution and spread it on the solid screening medium, culture it at 28°C for 3 days, pick a single colony and perform streak purification to obtain the primary screening strain; Step 2: Rescreening of strains The primary screening strains were inoculated into the seed culture medium, and cultured at 28°C and 150r / min for 1 day; 5% by volume of the inoculum was inoculated into the initial fermentation medium, and cultured at 28°C and 150r / min for 1 day, and then the enzyme activity was determined by chiral HPLC, and then the strains with high enzyme activity were selected and stored in 50vt% glycerol at 1 / 1 (v / v); Step 3: Colony characteristics Step 4: Molecular biological identification of the strain; extraction of genomic DNA, sequencing after PCR amplification, and sequencing comparison to determine the strain species; Step 5: Preservation of strains.

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