CN116814598A

CN116814598A - Phage lyase Lysrb8, coding gene thereof and application thereof

Info

Publication number: CN116814598A
Application number: CN202310709917.8A
Authority: CN
Inventors: 孙艳梅; 翁婧; 闫军军; 王卫富; 亢兴平; 杨巧丽; 王世伟
Original assignee: Shaanxi Huasen Shengbang Technology Co ltd; Shaanxi Biochemical Industry Technology Innovation Platform Co ltd
Current assignee: Shaanxi Huasen Shengbang Technology Co ltd; Shaanxi Biochemical Industry Technology Innovation Platform Co ltd
Priority date: 2023-06-15
Filing date: 2023-06-15
Publication date: 2023-09-29

Abstract

The invention relates to the field of genetic engineering, in particular to phage lyase Lysrb8, a coding gene thereof and application thereof, wherein the phage lyase is (a) or (b), and the phage lyase is formed by the sequence shown in SEQ ID NO:1, and a phage lyase consisting of the amino acid sequence shown in 1; (b) SEQ ID NO:1, or a protein derived from (a) in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:1 and/or a tag attached to the amino acid sequence of the amino acid sequence. The lyase provided by the invention has relatively stable protein, is hydrophilic protein, can inhibit growth of indicator bacteria ATCC7757, SRB-3 and SW-3, and can effectively lyse sulfate reducing bacteria.

Description

Phage lyase Lysrb8, coding gene thereof and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to phage lyase Lysrb8, a coding gene thereof and application thereof.

Background

Microbial corrosion (microbially induced corrosion, MIC) refers to corrosion of metals by the vital activities of various microorganisms and their metabolites, either directly or indirectly. Many microorganisms including bacteria, archaea and fungi have been found to cause corrosion, and bacteria associated with microbial corrosion are mainly iron bacteria (FB), sulfate-reducing bacteria (SRB), saprophytes (TGB), with corrosion in SRB being most severe.

SRB is an anaerobic microorganism which takes organic carbon source as electron donor and takes sulfur-containing oxides such as sulfate, sulfite, thiosulfate and the like as electron acceptors of organic matters to reduce the sulfur oxides into hydrogen sulfide under the anaerobic or partial anoxic condition. SRB has a corrosive effect on almost all common metallic materials such as steel, aluminum, zinc, copper, and alloying metals. The research of de Queiroz, et al, found that microorganisms easily form biofilms on metal surfaces, causing localized corrosion. Oil field recoveryIn water flooding, high concentrations of sulfate ions can cause massive SRB proliferation, producing H _z S, feS, severely corroding water injection lines and drilling equipment, a process also known as microbial acidizing of the oil field (microbial souring). Hydrogen sulfide accelerates the corrosion rate, pollutes crude oil, reduces crude oil quality, increases the subsequent processing cost, and in addition, has high toxicity, thereby causing serious threat to the lives of workers. The generated ferrous sulfate precipitate can cause the blockage of an oil layer so as to reduce the oil layer leakage rate, so that the exploitation difficulty is greatly increased. The acidification of the oil reservoir causes the normal production of the oil field to be jeopardized, resulting in huge economic loss. Metal corrosion causes economic losses of 2.5 trillion dollars each year worldwide, accounting for about 3.4% of the total worldwide production, while microbial corrosion (microbiallyinduced corrosion, MIC) accounts for 30% of it. MIC is mostly found in environments such as crude oil production, running water industry, fermentation workshops, etc., piping systems, and steel piles on wharfs, ports, and wharfs. Studies have shown that the petroleum industry in China costs hundreds of millions of dollars annually due to SRB corrosion. In addition, the heat exchange systems of the production pipeline systems, in particular of the power water systems, including the plants, are very severely corroded, and the resulting production losses are also great.

The SRB is generally inhibited by bactericides such as chlorine, formaldehyde, glutaraldehyde, quaternary ammonium salt and the like in oil fields, and the chemical bactericides have high sterilizing efficiency on bacteria and simple operation, but cause environmental pollution and harm to human health. Long-term use of bactericides can cause SRB to develop drug resistance. In recent years, it has been discovered that SRB can be killed or inhibited by the use of competition, antagonism and parasitism between microorganisms. The biological competition inhibiting technology has the characteristics of environmental friendliness, low price, large treatment range, simple operation and the like, but SRB grows in a pipeline to form a biological film, and the biological competition inhibiting technology has limited effects on SRB sessile bacteria growing in the biological film.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide phage lyase with high activity and high stability, and a coding gene and application thereof.

To achieve the above object, in a first aspect, the present invention provides a phage lyase shown in (a) or (b):

(a) Consists of SEQ ID NO:1, and a phage lyase consisting of the amino acid sequence shown in 1;

(b) SEQ ID NO:1, or a protein derived from (a) in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:1 and/or the amino acid sequence of the tag attached to the amino terminus and/or the carboxy terminus.

In a second aspect, the present invention provides a gene capable of encoding a phage lyase according to the first aspect.

In a third aspect, the present invention provides a recombinant vector comprising the gene according to the second aspect.

In a fourth aspect, the present invention provides a recombinant strain comprising the recombinant vector of the third aspect.

In a fifth aspect, the present invention provides a primer set for amplifying a gene as set forth in the second aspect above, the primer set comprising the nucleotide sequence set forth in SEQ ID NO:3 and the upstream primer shown in SEQ ID NO:4, and a downstream primer shown in FIG. 4.

In a sixth aspect, the present invention provides a method for preparing a phage lyase, the method comprising: (1) Culturing the recombinant strain of the fourth aspect, and inducing expression of a gene encoding a phage lyase; (2) isolating and purifying the expressed phage lyase.

In a seventh aspect, the present invention provides a composition comprising the phage lyase of the first aspect as an active ingredient.

In an eighth aspect, the invention provides a phage lyase according to the first aspect, a gene according to the second aspect, a recombinant vector according to the third aspect, a recombinant strain according to the fourth aspect, a primer set according to the fifth aspect and an application of the composition according to the seventh aspect in killing sulfate reducing bacteria, inhibiting growth of sulfate reducing bacteria and removing biofilms.

In a ninth aspect, the invention provides the phage lyase according to the first aspect, the gene according to the second aspect, the recombinant vector according to the third aspect, the recombinant strain according to the fourth aspect, the primer set according to the fifth aspect and the use of the composition according to the seventh aspect for alleviating, repairing or inhibiting reservoir acidification and/or metal pipeline corrosion.

1. The lyase provided by the invention (Lysrb 8 for short) has 311 amino acids in total, the molecular weight is about 36.66kDa, the isoelectric point is 4.87, the instability index is 27.68, the protein is stable, the average hydrophobicity value is-0.475, and the protease is hydrophilic protein.

2. The invention constructs a pET28a-Lysrb8 recombinant expression vector, and correctly expresses lyase Lysrb8 with the concentration of 0.4mg/mL.

3. Antibacterial activity studies have found that lyase Lysrb8 inhibits the growth of indicator bacteria ATCC7757, SRB-3 and SW-3, i.e., can effectively lyse sulfate-reducing bacteria.

4. The lyase provided by the invention can remove mature biofilm.

5. The lyase provided by the invention has higher thermal stability.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:

FIG. 1 shows the results of ProtScale prediction of Lysrb8 hydrophobicity.

FIG. 2 shows the purification results of recombinant proteins.

FIG. 3 shows a standard curve made from BSA protein standards.

FIG. 4 shows the result of protein specificity of Western blot detection.

FIG. 5 shows the lytic activity of the phage lytic enzyme provided by the invention.

FIG. 6 shows the cleavage spectrum of phage lyase provided by the present invention.

FIG. 7 shows the effect of phage lyase provided by the present invention on mature biofilm.

FIG. 8 shows the thermostability of phage lytic enzymes provided by the present invention.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In a first aspect, the present invention provides a phage lyase, which phage lyase is (a) or (b):

SEQ ID NO:1 as follows:

MLSLDDIDKEAVIWNLQRCGRIISETDRNMNGLSTNLGSLSKTNKYDSYNYTSNVNKCRYQYYEIQNNLKYIEMLIPLLKVYDGIDFGGFDTESIYSGFNRLKRGFTDLVSYGFKLAGGDILPSNMLNLMPRQMRDIINGLDDVKDTYEDYSGNQSSYSKFRQPTPSYNAVYPLNKTQETPGGHIFIQDDTDGAKLTMYKHPSGSVVYINDDGTFTIQSAKDMYRWADDDNLHVKGQVNIIIDGNANVDIGGNANVDIGGRGNIKTAGDLGMWGGNLTGEVDGVIDLHAKEGVSIWSEKGTNWKSGGG IDFDVFGNIRMKTTNGNIELN

in the present invention, the size of the term "enzyme activity" used, i.e., the size of the zone of inhibition of sulfate-reducing bacteria ATCC7757, is not stated in the contrary, specifically, the oxford cup method is adopted to detect the lytic activity of lyase Lysrb8, strain ATCC7757 is first cultivated to the logarithmic growth phase, 2 sterilized oxford cups are vertically placed on the solidified lower medium, then the prepared bacterial liquid and semi-solid medium are evenly mixed and poured on the upper layer, the oxford cups are taken out after solidification, and 100 μl of lytic enzyme is injected into the cup holes. Placing into an anaerobic tank, standing at 4deg.C for 4 hr, transferring to 37deg.C, culturing for 48 hr, and measuring the size of the inhibition zone.

In the present invention, the term "enzyme activity is unchanged" means that the sequence is not changed relative to the sequence as set forth in SEQ ID NO:1, and the enzyme activity of the derived lyase is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

The 20 amino acid residues that make up a protein can be divided into four classes according to side chain polarity: 1. nonpolar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (phe), tryptophan (Trp) and proline (Pro); 2. polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (gin), and tyrosine (Tyr); 3. positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "biochemistry" (second edition) handbook, shen Tong, wang Jingyan, pages 82-83, higher education Press, 12 months 1990). If amino acid residue substitutions of the same genus in proteins, such as Arg for Lys or Leu for IIe, are made, the residues do not change in the function of the protein domain (such as providing positive charge or forming a hydrophobic pocket structure) and therefore do not affect the steric structure of the protein, and thus the function of the protein can still be achieved. The substitution of the amino acid residues belonging to the same class may occur at any one of the amino acid residue positions of the phage lyase.

As described above, the phage lyase provided by the invention can be modified or mutated to obtain a derivative protein. The term "derivative protein" as used herein refers to a protein having an amino acid sequence different from that of a phage lyase having the above amino acid sequence, or may have a modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants may be obtained by various techniques such as random mutagenesis by irradiation or mutagens and the like, or by techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derivatized proteins" also include analogs having residues of the natural L-type amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta-amino acids, y-amino acids, etc.).

The modified (typically without altering the primary structure, i.e., without altering the amino acid sequence) forms include: chemically derivatized forms of proteins such as acetylated or carboxylated in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.

For ease of purification, the (a) may also be modified with a tag common in the art, for example, (b) may be obtained by ligating a tag shown in Table 1 below (such as at least one of Poly-Arg, poly-His, FLAG, strep-tag II and c-myc) at the amino-and/or carboxy-terminus of (a). The label does not affect the activity of the phage lyase of the invention, and whether the label is added or not can be selected according to the requirement in the actual application process.

TABLE 1

Label (Label)	Residue number	Amino acid sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The phage lyase can be obtained by artificial synthesis, or can be obtained by synthesizing the encoding gene and then biologically expressing.

In a second aspect, the present invention also provides a gene capable of encoding the phage lyase described above.

Accordingly, the gene may be (1) or (2) as follows:

(1) The nucleotide sequence is shown in SEQ ID NO:2, a DNA molecule shown in fig. 2;

(2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (1) and which encodes a phage lyase with unchanged enzymatic activity. In some embodiments, the stringent conditions may be: hybridization was performed in a solution of 6 XSCC, O.5% SDS at 65℃and then the membranes were washed once with 2 XSCC, 0.1% SDS and 1 XSCC, 0.1% SDS.

In the present invention, the constant enzyme activity means that the percentage (relative activity) between the enzyme activity of the protein encoded by (2) and the enzyme activity of the protein encoded by (1) is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%) under the same measurement conditions.

It is well known in the art that of the 20 different amino acids that make up a protein, other than Met (ATG) or Trp (TGG) are each encoded by a single codon, the 18 other amino acids are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989, see page 950 appendix D). That is, due to the degeneracy of the genetic code, the nucleotide sequence of the gene encoding the same protein may differ, since the substitution of the third nucleotide in the triplet codon, which determines most of the codons of one amino acid, does not change the composition of the amino acid. The nucleotide sequences of genes encoding them can be deduced entirely from the amino acid sequences disclosed in the present invention and the amino acid sequences obtained from the amino acid sequences, which are not changed in the phage lyase activity, according to known codon tables, obtained by biological methods (e.g., PCR methods, mutation methods) or chemical synthesis methods, and thus are included in the scope of the present invention. Conversely, by using the DNA sequences disclosed herein, amino acid sequences consistent with the phage lyase activity of the invention can also be obtained by modifying the nucleic acid sequences provided herein by methods well known in the art, such as the method of Sambrook et al (molecular cloning, cold spring harbor laboratory Press, new York, U.S. second edition, 1989).

In a preferred embodiment, the nucleotide sequence of the gene is set forth in SEQ ID NO: 2.

SEQ ID NO：2：

ATGCTGTCGCTTGATGATATCGACAAAGAAGCTGTCATTTGGAATCTGCAACGTTGTGGACGCATCATCTCCGAGACAGACCGCAACATGAATGGTCTGTCAACGAATCTTGGGTCTTTGTCCAAAACAAACAAATACGATTCATACAACTACACAAGCAATGTCAATAAGTGTCGATACCAATACTATGAGATTCAAAACAATCTCAAGTATATTGAAATGCTTATACCATTGCTCAAAGTATATGACGGAATAGACTTTGGTGGGTTTGACACAGAAAGCATCTATAGTGGTTTCAACCGCCTGAAAAGAGGCTTCACAGATTTGGTCTCATACGGGTTCAAGCTTGCTGGTGGTGATATCCTGCCGTCAAACATGTTGAATCTCATGCCAAGACAAATGAGAGACATTATCAATGGTCTGGATGATGTAAAAGACACATACGAAGACTACAGTGGAAATCAATCGTCCTATAGTAAGTTCCGTCAACCAACTCCATCTTACAACGCTGTCTATCCACTCAACAAAACACAAGAGACTCCCGGTGGTCACATTTTCATTCAAGACGACACAGATGGTGCAAAGCTGACCATGTACAAGCATCCTTCGGGGTCAGTCGTCTACATCAATGACGATGGGACTTTCACAATACAGTCAGCTAAAGATATGTATAGGGTTGTTGCAGACGACGACAACTTGCATGTCAAAGGTCAAGTCAACATCATCATTGACGGAAATGCAAATGTTGACATCGGTGGAAATGCAAATGTTGACATCGGTGGAAGAGGCAACATCAAGACCGCTGGCGATTTGGGAATGGTTGTTGGTGGAAATCTCACCGGGGAAGTCGATGGTGTGATTGACCTACATGCAAAAGAAGGTGTCTCAATCTGGTCTGAAAAGGGAACCAACTGGAAATCAGGTGGCGGCATAGATTTTGACGTTTTGGCAACATCCGCATGAAGACCACCAATGGCAACATCGAACTCAACTAA

As described above, the 5 'and/or 3' end of the nucleotide sequence may be linked to the coding sequence of the tag shown in Table 1 above, accordingly.

The nucleotide sequence provided by the invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombination method or an artificial synthesis method. For example, templates and primers can be readily obtained by those skilled in the art from the nucleotide sequences provided herein, and the relevant sequences can be obtained by amplification using PCR.

Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombinant methods. The obtained nucleotide sequence is cloned into a vector, transferred into genetically engineered bacteria, and then separated from the proliferated host cells by a conventional method to obtain the related nucleotide sequence.

In addition, the nucleotide sequence of interest can be synthesized by well-known methods of artificial chemical synthesis.

In a third aspect, the present invention provides a recombinant vector comprising the gene of the present invention.

As the "vector" used for the recombinant vector, various vectors known in the art, such as various plasmids, cosmids, phages, retroviruses and the like, are used, and the pET28a plasmid is preferred in the present invention. The recombinant vector may be constructed by using various endonucleases capable of having cleavage sites at the vector multiple cloning sites (e.g., sal I, bamH I, ecoR I, etc. for pUC 18; nde I, nhe I, ecoR I, bamH I, hind III, xho I, etc. for pET28 a) to obtain a linear plasmid, and ligating the gene fragments cut with the same endonucleases. The invention preferably adopts Nhe I and Xho I double-enzyme cutting pET28a and gene fragments connected with the same, and the recombinant vector is constructed by connecting the gene fragments through ligase.

In a fourth aspect, the present invention provides a recombinant strain comprising the recombinant vector provided herein.

The recombinant vector may be transformed, transduced or transfected into a host cell (strain) by methods conventional in the art, such as calcium chloride chemical transformation, high voltage shock transformation, preferably shock transformation. The host cell may be a prokaryotic or eukaryotic cell, preferably a coryneform bacterium such as Escherichia coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis) or a yeast such as Pichia pastoris (Pichia pastoris) or Saccharomyces cerevisiae (Saccharomyces cerevisiae), more preferably the host cell is Escherichia coli such as Escherichia coli BL21 (DE 3) or Escherichia coli DH 5. Alpha.

In a fifth aspect, the invention provides a primer set for amplifying a gene as set forth in the second aspect above, the primer set comprising the nucleotide sequence set forth in SEQ ID NO:3 and the upstream primer shown in SEQ ID NO:4, and a downstream primer shown in FIG. 4.

The primer group provided by the invention can specifically amplify the genes shown in the second aspect. The conditions and systems of the amplification may employ those conventional in the art.

The culture conditions are conventional ones, such as those using LB medium (solventIs water, and the solute and the final concentration thereof are respectively: tryptone 10g/L, yeast extract 5g/L, naCl 10 g/L), at 35-37deg.C to OD ₆₀₀ 0.6. Because the recombinant strain provided by the invention contains the gene for encoding phage lyase, phage lyase can be efficiently expressed. After culturing, separating and purifying to obtain the phage lyase with high purity. Separation and purification (e.g., isopropyl-beta-d-thiogalactoside (IPTG) is added to the culture broth to a final concentration of 0.15-0.25mm, and the culture broth is subjected to shaking culture at 12-18 ℃ for 8-12 hours, then the cells are collected, suspended in PBS buffer, sonicated, and purified to obtain phage lyase) can be performed by methods well known to those skilled in the art, and will not be described herein.

In some embodiments, the phage lyase is present in an amount of 10-90 wt.%, based on the total weight of the composition. The composition may further comprise solvents (such as glycerol, saccharide and protease inhibitor, etc. protein protectant), agonists (such as NiCl) ₂ Calcium chloride), and the like.

In an eighth aspect, the invention also provides applications of the phage lyase, the gene, the recombinant vector, the recombinant strain, the primer and the composition in killing sulfate reducing bacteria, inhibiting growth of the sulfate reducing bacteria and removing biofilms.

The phage lyase provided by the invention can cleave SRB (sulfate-reducing bacteria sulfate-reducing bacteria) so as to achieve the aim of killing and/or inhibiting the SRB.

The phage lyase provided by the invention can remove mature biofilm formed by SRB. In some embodiments, the "mature biofilm" is obtained by: the SRB bacterial liquid is evenly mixed with the culture medium according to the proportion of 1:4. 100 mu L of bacterial liquid is sucked up and added into a 96-well plate, and the mixture is placed into an anaerobic tank to be cultured for 48 hours in a constant temperature incubator at 30 ℃. In some embodiments, a "mature biofilm" refers to a stable biofilm formed by an SRB in an environment in which it is located.

In some embodiments of the invention, the phage lyase or the composition of the invention may be applied directly to the surface of an environment containing SRB. Examples of such solid state environments may include, but are not limited to, metal equipment underground piping for sewage treatment plants, metal equipment for oilfield exploitation, metal equipment for tap water industry, metal equipment for wharfs, ports, metal equipment for fermentation plants. In some embodiments, the SRB-containing environment is in a liquid state, and the phage of the invention as described above or the bactericidal composition as described above may be added directly to the liquid phase containing the SRB. Examples of SRBs containing environments that are liquid include, but are not limited to, mine wastewater, hydrocarbon reservoirs, coal reservoirs, and industrial piping systems.

In some embodiments, the reservoir acidification and/or metal corrosion is caused by sulfate-reducing bacteria.

In the present invention, the environment to be relieved, repaired or inhibited from corrosion by sulfate-reducing bacteria is generally a metal surface on which SRBs are grown, for example, various metal piping systems and the like, surfaces of metal equipment and the like.

The present invention will be described in detail by examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

1) Preparation of culture Medium and solution

(1) LB medium (1L): 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride are weighed. The solid LB culture medium is added with 1.2g/100mL of agar powder.

(2) SOC Medium (1L): weighing tryptone20.0g, yeast extract 5.0g, sodium chloride 0.6g, were added to 950mL deionized water to dissolve them sufficiently, and then added to a final concentration of 10mM MgCl, respectively ₂ ，10mM MgSO ₄ 20mM D-glucose, pH 7.5, and finally volume to 1L, autoclaved at 115℃for 20 minutes.

(3) Kanamycin (10 mg/mL): 0.1g of kanamycin powder is weighed, the volume is fixed in 10mL by deionized water, the powder is filtered by a 0.22 mu m filter, the working concentration is 50 mu g/mL,

(4) IPTG (1 mol/L): 2.38g of the powder is weighed and dissolved in a small amount of deionized water, the volume is finally fixed to 10mL, and the powder is filtered by a 0.22 mu m filter and stored at the temperature of minus 20 ℃ in a dark place, wherein the working concentration is 0.2mmol/L.

(5) 10% SDS: 10.0g of SDS was weighed, dissolved in about 70mL of deionized water, pH was adjusted to 7.2 using concentrated hydrochloric acid, and finally the volume was set to 100mL.

(6) 30% acrylamide: bisacrylamide 1.0g,Acrylamide 29.0g was weighed separately, added to deionized water to a volume of 100mL, filtered through a 0.45 μm filter membrane and stored at 4deg.C in the absence of light.

(7) 10% ammonium persulfate (10% ap): 0.1g of ammonium persulfate was weighed and dissolved in 1.0mL of sterilized deionized water and prepared for use.

(8) Coomassie mL, blue R250 staining: 1.0g of Coomassie brilliant blue is weighed, 100mL of glacial acetic acid and 250mL of isopropanol are measured and stirred uniformly, deionized water is used for constant volume to 1L, and after particulate matters are removed by filter paper, the filter paper is preserved at room temperature for standby.

(9) PAGE gel decolorization solution: 100mL of glacial acetic acid and 50mL of ethanol are respectively measured, deionized water is added for constant dissolution to 1L, and the mixture is stirred uniformly and stored at room temperature for standby.

EXAMPLE 1 analysis of physicochemical Properties of Lysrb8

Using Expasy @https://www.expasy.org/) ProtScale and ProtParam tools in the protein analysis System analyze the molecular weight, stability, isoelectric point, hydrophobicity, etc. of the lyase gene Lysrb 8.

The amino acid sequence of Lysrb8 is as follows SEQ IN NO:1, lysrb8 shares amino acid 331 (Table 6), with a molecular weight of approximately 36.66kDa, an isoelectric Point (PI) of 4.87, a total number of negatively charged residues of 43, a total number of positively charged residues of 32, and an instability index of 27.68 (< 40), thus judging that the protein is relatively stable. The result of predicting the hydrophobicity of Lysrb8 by ProtScale is shown in FIG. 1, the hydrophobicity of Lysrb8 is maximum 1.767, the minimum-2.489 and the average hydrophobicity-0.475, and thus the protein is a hydrophilic protein.

TABLE 6 Lysrb8 amino acid composition

The conserved domain encoded by amino acids 161-327 of the Lysrb8 gene was found to be 5super family using CD-Search in NCBI database. Members of this family include baseplate hub subunit (substrate center subunit) and tail lysozyme, which hydrolyzes bacterial cell walls.

Example 2 expression of Lysrb8

1) Amplification of target Gene

The upstream and downstream primers were designed based on the nucleotide sequence of the lyase Lysrb8 (Table 2), and the target gene was amplified by PCR using the synthesized primers, using the genome of phage SRB7757 (CGMCC 45383) as a template. The PCR reaction system is shown in Table 3, and the reaction procedure is 94℃for 30s,98℃for 10s,55℃for 30s,72℃for 1min,72℃for 2min, and 34 cycles. The PCR reaction product is verified by agarose gel electrophoresis, and finally whether the target band is amplified or not is observed under a gel imager. If the target band is amplified, the PCR product is purified and recovered by using a DNA purification and recovery kit.

TABLE 2 primers

TABLE 3PCR reaction System

2) Enzyme digestion of the Gene and plasmid of interest

The vector pET-28a was digested with the restriction enzymes BamH I and Xhol I, and the digestion reaction system was shown in Table 4, and digested at 37℃for about 10 hours. After the reaction is finished, the enzyme digestion product is subjected to electrophoresis identification, purification and recovery.

Table 4 enzyme digestion System

3) Ligation and transformation

The digested PCR products and plasmids were mixed according to the reaction system in Table 5, and then placed in a metal bath at 16℃for 8-12 hours. The recombinant plasmid is transformed into DH5 alpha competence by adopting a heat shock method, and the specific operation is as follows: firstly, adding 5 mu l of the connection product into 50 mu l of melted DH5 alpha, blowing and uniformly mixing, and then carrying out ice bath for 30min; after the competence is recovered, placing the mixture in a metal bath for heat shock for 90s at 42 ℃, and immediately taking out the mixture and placing the mixture on ice for 3min; then adding 700 mu l of SOC liquid culture medium into the centrifuge tube, and after uniformly mixing, propagating thalli for 1h at 37 ℃ and 200 r/mm; and finally, centrifuging for 2min at 4000r/min in a centrifuge, collecting and re-suspending the thalli, coating the thalli on LB plates with Kan resistance, culturing for 12h at 37 ℃, and observing the growth condition of colonies.

TABLE 5 ligation reaction System

4) Identification of recombinant plasmids

Randomly picking 4 single bacterial colonies from the flat plate, coating the single bacterial colonies on a new LB flat plate containing Kan resistance, and after bacterial colonies grow out, picking the single bacterial colonies to be inoculated in an LB liquid culture medium containing Kan resistance, and culturing for 12 hours at 37 ℃ at 150 r/min. Then extracting recombinant plasmid, carrying out PCR and sequencing verification on the extracted plasmid,

5) Transformation of recombinant plasmids

Transfer of the identified correct recombinant plasmid into BL21 competent cells by heat shock method, and the specific procedure is the same as in step 3).

6) Prokaryotic expression of lyase genes

a) The BL21 expression strain which is successfully transformed is picked up for monoclonal, inoculated into LB liquid medium (3 mL) with Kan concentration of 50 mug/mL, and cultured at 37 ℃ under shaking of 200r/min for overnight.

b) Then, according to 1:100 the above culture broth was inoculated into 50. Mu.g/mL Kan LB liquid medium (30 mL), and cultured at 37℃for 200r/min until the growth log phase (OD ₆₀₀ ＝0.6--0.8)。

c) 1mL of the above culture broth was aspirated, centrifuged at 10000r/min for 2min, and the bacterial pellet was resuspended in 100. Mu.L of 1X SDS Loading Buffer as a pre-induction control.

d) IPTG was added to the remaining culture medium at a final concentration of 0.2mM, and the mixture was cultured at 15℃and 220r/min overnight to induce expression of the fusion protein.

e) 1ml of the induced solution was taken out, centrifuged at 10000r/min for 2min, and then the bacterial pellet was resuspended with 100. Mu.l of 1X SDS Loading Buffer as a post-induction sample. The remaining induced solution was centrifuged at 4000r/min for 10min and the bacterial pellet was resuspended in PBS buffer.

f) The resuspension was subjected to ultrasonic disruption, and then the supernatant and the precipitate were each added to 1X SDS Loading Buffer for resuspension.

g) The 12% SDS-PAGE protein pre-gel is placed in an electrophoresis tank, poured into 1 XTris-Gly electrophoresis buffer, 80. Mu.l of sample is taken, 20. Mu.l of 5 Xbuffer (containing B-mercaptoethanol) is added, the mixture is evenly mixed and boiled for 10 minutes, 5. Mu.l of the mixture is taken after centrifugation and added into a gel well, and Maker is added into one air for electrophoresis. Then stained with coomassie brilliant blue for 30min, and the decolorized solution was repeatedly decolorized until protein bands were visible, and observed under a gel imager and recorded by photographing.

7) Purification of recombinant proteins

Purifying the protein solution obtained in the above steps by using a Ni column to obtain a relatively purified target protein, and eluting the target protein by using a Ni-IDA Binding-Buffer balancing column. The collected protein eluate was added to a dialysis bag, dialyzed overnight against PBS and analyzed by 12% SDS-PAGE.

As a result, as shown in FIG. 2, it was revealed from FIG. 2 that the eluate after purification by Ni column contained a single protein band with little impurity band, which indicated that a Lysrb8 protein of higher purity was obtained (note FIG. 2A: lane 1: pET28a no load; lane 2: pre-induction sample; lane 3: post-induction sample; lane 4: supernatant after induction disruption; lane 5: precipitation after induction disruption; note FIG. 2B: post-disruption treatment sample; lane 2: effluent sample; lanes 3 and 4: elution sample).

Test example 1

1) Recombinant protein concentration determination

The dissolved BSA protein standard is diluted to the standard sample with the concentration of 0.1mg/mL, 0.2mg/mL, 0.A mg/mL, 0.6mg/mL, 0.8mg/mL and 1.0mg/mL, the absorbance at 595nm is measured by an ultraviolet spectrophotometer, and a standard curve is drawn. The absorbance of the recombinant protein solution at 595nm was then measured and its concentration was calculated by a standard curve.

As a result, as shown in FIG. 3, the absorbance of the purified Lysrb8 at OD595 was 0.182, respectively, and the concentration of the Lysrb8 was calculated to be about 0.4mg/mL.

2) Western blot detection protein specificity

Electrophoresis: the recombinant protein samples were subjected to SDS-PAGE.

Transferring: soaking the SDS-PAGE gel after electrophoresis in a membrane transferring liquid for 5-10min, taking a PVDF membrane with proper size, soaking the PVDF membrane in methanol for 15s until the membrane becomes semitransparent, washing the PVDF membrane with ultrapure water, and then placing the PVDF membrane into the membrane transferring liquid for membrane transferring, soaking for a plurality of minutes and then transferring the membrane. The film transfer sequence is as follows: negative electrode-foam-sponge cushion-three layers of filter paper-SDS-PAGE gel-PVDF membrane-three layers of filter paper-foam cushion-positive electrode. The current was adjusted to 280mA and the film was transferred for 1 hour.

Closing: after the end of the electrotransfer, the PVDF membrane was removed and washed rapidly with PBST for 4 times, each for 5min. Then the mixture was placed on a 37℃shaker to be blocked for 1 hour.

Incubation resistance: PBST was diluted with primary antibody (1:4000), and the dilution volume was typically 3-4ml in the antibody incubation cassette overnight at 4 degrees.

Washing the film: PVDF membrane was removed from primary antibody and washed rapidly with PBST 4 times for 5min each.

Secondary antibody incubation: the secondary antibody was diluted 1:20000 with 5% milk and the washed PVDF membrane was incubated in the secondary antibody for 1h at 37 ℃.

Washing the film: and (5) washing the membrane again, wherein the step is the same as the step (5).

Developing: the ECL method is adopted, firstly, flat paper is used for absorbing water on the PVDF film, the film is placed on a plane, the solution A and the solution B are uniformly mixed in equal proportion and then added on the film for reaction for 1min, then the film is taken out, ECL luminous liquid is discarded, and the film is developed in a cassette, photographed and recorded.

As a result, as shown in FIG. 4, a specific band was seen at around 40kDa in Lysrb8, and the size of the cleavage enzyme protein molecule was expressed correctly.

3) Detection of cleavage Activity

The lytic activity of the lyase Lysrb8 is detected by adopting an oxford cup method, firstly, the strain ATCC7757 is cultivated to a growth logarithmic phase, 2 sterilized oxford cups are vertically placed on a solidified lower layer culture medium to ensure no gap between the cup bottom and the culture medium, then the prepared bacterial liquid and the semisolid culture medium are uniformly mixed and poured into an upper layer, the oxford cups are taken out after solidification, 100 mu L of lyase is taken and injected into cup holes, and PBS buffer is additionally added for comparison. Placing into an anaerobic tank, standing at 4deg.C for 4 hr, transferring to 37deg.C, culturing for 48 hr, and observing whether there is a zone of inhibition.

As a result, as shown in FIG. 5, it can be seen that Lysrb8 can produce a transparent zone of inhibition, and thus Lysrb8 can be judged to be active.

4) Determination of cleavage Spectrum of recombinant proteins

The 11 strains in Table 7 were activated to determine the cleavage spectrum of the lyase Lysrb8, the bacteria were plated by a double-layer plate method, 10. Mu.L of the lyase Lysrb8 was added dropwise to each plate, 10. Mu.L of the lysate Lysrb8 was added dropwise simultaneously, and a negative and positive control was obtained by sterilization and preservation, and the presence or absence of a zone of inhibition was observed in the anaerobic tank at 37 ℃.

As a result, as shown in FIG. 6, it was found that Lysrb8, a cleavage enzyme, was able to cleave ATCC7757, and a transparent zone of inhibition was formed on both the surfaces of SRB-3 and SW-3, indicating a broader cleavage spectrum of Lysrb 8.

Test example 2 Effect of phage lyase on mature biofilm

The polyvinyl chloride 96-well plate is put into a biological safety cabinet in advance, and ultraviolet irradiation is carried out for 30min. The bacterial liquid is evenly mixed with the culture medium according to the proportion of 1:4. 100 mu L of bacterial liquid is sucked up and added into a 96-well plate, and the mixture is placed into an anaerobic tank to be cultured for 48 hours in a constant temperature incubator at 30 ℃. After the cultivation is completed, the liquid is sucked out by a discharge gun, the liquid is washed three times by PBS, and the water in the hole is sucked clean in the last washing. The 120. Mu.LPBS was aspirated separately and a concentration of 100. Mu.g/mL of lyase was added to the 96-well plate. Placing into an anaerobic tank, placing into a constant temperature incubator at 30 ℃, and standing for culturing for 6 hours. After the liquid was sucked out by a discharge gun, and 200. Mu.L of water was sucked into a waste liquid tank to perform three times of washing, 150. Mu.L of 0.1% crystal violet solution was added, and after standing at room temperature for 5 minutes, it was sucked out. After washing, 160. Mu.L of 30% acetic acid solution was added thereto, and the mixture was allowed to stand at room temperature for 10 minutes, followed by measurement of absorbance at 595nm using a microplate reader.

Results As shown in FIG. 7, ATCC7757 biofilm was significantly reduced after incubation with Lysrb8 at 100. Mu.g/mL. Lysrb8 treatment reduced biofilm by about 62.2% compared to the control group. The above shows that Lysrb8 has a scavenging effect on the mature biofilm of sulfate-reducing bacterium ATCC 7757.

Test example 3 cleavage enzyme optimum action temperature test

And (3) taking 500mL of bacterial liquid cultured to a growth log phase, centrifuging and collecting the precipitate, and finally, re-suspending the bacterial precipitate by using 20mL of PBS solution. The liquid is placed in an ultrasonic cell disruption instrument for disruption for 130min with a disruption power of 35%, disruption for 3s and intermittent for 4s. The disrupted bacterial liquid was centrifuged at 7000r/min for 20min, and the supernatant was discarded, and 50mL of PBS solution was added to the pellet and the pellet was blown and mixed well. At this time, cell walls of the host bacteria are cell debris, and serve as reaction substrates for the following experiments.

The reaction temperature was set at 30℃and 37℃and 50℃and 60℃and 70℃respectively, and the cleavage enzyme was added to the reaction substrate, the final concentration of the cleavage enzyme was controlled at 100. Mu.g/mL, and the reaction was carried out at different temperatures for 30 minutes, and the control group was substituted with PBS buffer. Absorbance was measured at a wavelength of 600nm, three in each group.

As a result, the relative activities of the lyase at different temperatures were different as shown in FIG. 8. The test result shows that the lyase Lysrb8 still has activity at 70 ℃ and has good action effect, which indicates that the lyase Lysrb8 has good tolerance to high temperature.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims

1. A phage lyase, wherein said phage lyase is (a) or (b):

2. A gene capable of encoding the phage lyase according to claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is set forth in SEQ ID NO: 2.

4. A recombinant vector comprising the gene of claim 2 or 3.

5. A recombinant strain comprising the recombinant vector of claim 4.

6. A primer set for amplifying a gene as set forth in claim 2 or 3, characterized in that the primer set comprises the sequence set forth in SEQ ID NO:3 and the upstream primer shown in SEQ ID NO:4, and a downstream primer shown in FIG. 4.

7. A method of preparing a phage lyase, comprising the steps of:

(1) Culturing the recombinant strain of claim 5 or 6, inducing expression of a gene encoding a phage lyase;

(2) Separating and purifying the expressed phage lyase.

8. A composition comprising the phage lyase according to claim 1 as an active ingredient.

9. Use of the phage lyase of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4, the recombinant strain of claim 5, the primer set of claim 6, and the composition of claim 8 for killing sulfate-reducing bacteria, inhibiting growth of sulfate-reducing bacteria, and removing biofilm.

10. Use of the phage lyase of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4, the recombinant strain of claim 5, the primer set of claim 6, and the composition of claim 8 for alleviating, repairing or inhibiting reservoir acidification and/or metal pipeline corrosion.