WO2022111301A1 - 一种嵌合溶菌酶变体及其在动物饲料添加剂中的应用 - Google Patents

一种嵌合溶菌酶变体及其在动物饲料添加剂中的应用 Download PDF

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WO2022111301A1
WO2022111301A1 PCT/CN2021/130202 CN2021130202W WO2022111301A1 WO 2022111301 A1 WO2022111301 A1 WO 2022111301A1 CN 2021130202 W CN2021130202 W CN 2021130202W WO 2022111301 A1 WO2022111301 A1 WO 2022111301A1
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lysozyme
chimeric
animal feed
variant
lysozyme variant
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PCT/CN2021/130202
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French (fr)
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郑斐
严婷
朱继东
徐红
孙艳
刘延杰
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南京百斯杰生物工程有限公司
济南百斯杰生物工程有限公司
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Priority to US18/254,428 priority Critical patent/US20240093171A1/en
Priority to CN202180079538.4A priority patent/CN116507356A/zh
Publication of WO2022111301A1 publication Critical patent/WO2022111301A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of bioengineering, and relates to a chimeric lysozyme variant and its application in animal feed additives.
  • Lysozyme (EC 3.2.1.17) is a hydrolase that acts on the cell wall of microorganisms, also known as muramidase. It can effectively hydrolyze the peptidoglycan of bacterial cell wall, and its mechanism is mainly through the hydrolysis of ⁇ - between N-acetylmuramic acid and N-acetylglucosamine.
  • the 1,4 glycosidic bond breaks the peptidoglycan backbone and causes the cell wall to rupture, eventually leading to bacterial lysis.
  • Lysozyme occurs naturally in many organisms, such as viruses, plants, insects, birds, reptiles, and mammals. In mammals, lysozyme has been isolated from nasal secretions, saliva, tears, intestinal contents, urine and milk. Lysozymes have been classified into seven different families of glycoside hydrolases (CAZy www.cazy.org): GH18, GH19, egg white lysozyme (GH22), goose egg white lysozyme (GH23), bacteriophage T4 lysozyme (GH24) ), Sphingomonas flagellin (GH73), and Chalaropsis lysozyme (GH25). Lysozyme is a non-toxic protein with no side effects to humans and mammals.
  • lysozyme as a non-specific immune factor, has an important application prospect in the replacement of antibodies. Lysozyme can catalyze the hydrolysis of the ⁇ -1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bacterial cell wall, resulting in the exudation of bacterial contents and the dissolution of bacteria, thereby achieving antibacterial and other effects .
  • lysozyme is used in combination with polymeric phosphate and glycine, which has a good antiseptic effect. Adding lysozyme to the feed can prevent mildew, prolong the storage period of the feed, and reduce unnecessary losses. The combined use of lysozyme and glucose oxidase also has a synergistic effect.
  • an object of the present invention is to provide a chimeric lysozyme variant with improved heat resistance and a polynucleotide encoding the variant, and a new chimeric lysozyme variant sequence is constructed.
  • the thermostability of the chimeric lysozyme variant at 60°C is nearly doubled compared to the parental sequence;
  • another object of the present invention is to provide a thermostable lysozyme variant.
  • the application of the improved chimeric lysozyme variant in an animal feed additive the feed additive has a significant effect on animal growth performance, and greatly improves the conversion rate of feed.
  • the present invention provides a chimeric lysozyme variant, the amino acid sequence of the chimeric lysozyme variant is as shown in SEQ ID NO: 8, or has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% The above sequence identity; and the chimeric lysozyme variant has lysozyme activity.
  • the amino acid sequence is shown in SEQ ID NO: 8, that is, the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 6
  • the first 109 amino acids at the N-terminus are replaced by SEQ ID NO: 6
  • the first 111 amino acids of the N-terminal of the amino acid sequence shown in ID NO: 4 are obtained.
  • the present invention also provides the application of the chimeric lysozyme variant in the preparation of an alternative antibiotic product.
  • substitute antibiotic products refers to products that can replace antibiotics, which can be used in animal feed additives, as a potential substitute for antibiotics, to inhibit the reproduction of harmful microorganisms, protect the intestinal health of animals, and improve animal health. immunity, etc.
  • the present invention also provides the use of the chimeric lysozyme variant as described above, which:
  • the present invention also provides a method for improving the nutritional value of animal feed, to which the chimeric lysozyme variant as described above is added.
  • the present invention also provides an animal feed additive comprising the chimeric lysozyme variant as described above.
  • the feed additive further comprises one or more components selected from the group consisting of:
  • the vitamins include fat-soluble vitamins and water-soluble vitamins; non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3; non-limiting examples of water-soluble vitamins include vitamin C, vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate such as Ca-D-pantothenate.
  • Non-limiting examples of such minerals include calcium, magnesium, phosphorus, potassium, sodium, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc.
  • Non-limiting examples of such amino acids are lysine, alanine, beta-alanine, threonine, methionine, and tryptophan.
  • the phytobiotics are a group of natural growth promoters or non-antibiotic growth promoters from herbs, spices or other plants used as feed additives.
  • Phytobiotics can be single substances prepared from essential oils/extracts, single plants and plant mixtures (herbal products) or mixtures of essential oils/extracts/plants (specialty products). Examples of phytobiotics are rosemary, sage, oregano, thyme, cloves and lemongrass.
  • essential oils are thymol, eugenol, m-cresol, vanillin, salicylates, resorcinol, guajacol, gingerol, lavender oil, ionone, iridone, Eucalyptol, Menthol, Peppermint Oil, Alpha-Pinene, Limonene, Anethole, Linalool, Methyl Dihydrojasmonate, Carvacrol, Propionate/Propionate, Acetic Acid/Acetate, Butyl acid/butyrate, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/or benzyl salicylate, cinnamaldehyde, vegetable polyphenols (tannins), Turmeric and Turmeric Extract.
  • the prebiotics are substances that induce the growth or activity of microorganisms (eg, bacteria and fungi) that contribute to the health of the host.
  • Prebiotics are generally indigestible fibrous compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of beneficial bacteria that colonize the large intestine by serving as their substrates.
  • prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the gastrointestinal (GI) tract.
  • Yeast derivatives inactivated whole yeast or yeast cell walls
  • They typically include mannooligosaccharides, yeast beta-glucans, or protein content, and are typically derived from the cell wall of yeast (Saccharomyces cerevisiae).
  • organic acids are widely distributed in nature as normal constituents of plant or animal tissues. They are also formed by microbial fermentation of carbohydrates mainly in the large intestine. They are often used as an alternative to antibiotic growth promoters in pig and poultry production because of their preventive effect on intestinal problems such as necrotizing enteritis in chickens and E. coli infection in piglets.
  • Organic acids can be sold as a single component or usually as a mixture of 2 or 3 different organic acids. Examples of organic acids are propionic, formic, citric, lactic, sorbic, malic, acetic, fumaric, benzoic, butyric and tartaric acids or their salts (usually sodium or potassium salts such as potassium diformate or sodium butyrate).
  • the feed additive of the present invention may further comprise colorants, stabilizers, growth improving additives and aroma compounds/flavors, polyunsaturated fatty acids (PUFA), reactive oxygen species generating substances, antioxidants, antimicrobial peptides, antifungal polypeptides, and mycotoxins Control compounds.
  • PUFA polyunsaturated fatty acids
  • reactive oxygen species generating substances antioxidants, antimicrobial peptides, antifungal polypeptides, and mycotoxins Control compounds.
  • the added amount of the chimeric lysozyme variant is 100-1000 g (100-1000 g/t) of the chimeric lysozyme variant per ton of animal feed; preferably, the added amount is per ton of animal
  • the chimeric lysozyme variant was added to the feed at 250-500 g (250-500 g/t).
  • the present invention also provides an animal feed containing the above-mentioned animal feed additive.
  • the feed also includes a basal diet.
  • the basic diet was prepared according to different growth stages with reference to NRC (1994) Chicken Nutritional Requirements, Chinese Chicken Breeding Standards (2004), and Chinese Feed Composition and Nutritional Value Table (2020).
  • the added amount of the animal feed additive is 250-500 g (250-500 g/t) of the animal feed additive per ton of animal feed.
  • Lysozyme itself is a natural protein, non-toxic, and is a highly safe feed additive. In the absence of any antibiotics, adding feed lysozyme preparation to the feed can promote the digestion and absorption of nutrients in the feed, improve the animal weight gain and feed conversion rate, and reduce animal morbidity and mortality.
  • the present invention also provides a polynucleotide encoding the chimeric lysozyme variant according to any one of the above.
  • nucleotide sequence of the polynucleotide is shown in SEQ ID NO:7.
  • the present invention also provides a recombinant vector containing a polynucleotide encoding a chimeric lysozyme variant as described above.
  • the present invention also provides host cells containing the recombinant vectors as described above.
  • the host cell can be any one of Aspergillus niger, Pichia pastoris, Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis or Escherichia coli, the present invention does not specifically limit the type of host cell,
  • the chimeric lysozyme variant of the present invention can be successfully expressed by conventional experimental methods.
  • the invention also provides a method for culturing a lysozyme recombinant expression strain of Aspergillus niger in a shake flask.
  • the lysozyme positive transformant is inoculated into a shake flask containing a YPM medium, cultured on a shaker bed, and the supernatant of the fermentation liquid is collected by centrifugation, and assayed. Lysozyme activity.
  • composition and content of the YPM medium are: yeast extract 0.2%, peptone 0.2%, maltose 2%.
  • the temperature of the shaker culture is 30-35°C; preferably, it is 34°C.
  • the rotating speed of the shaker culture is 180-250 rpm; preferably, it is 220 rpm.
  • the time of the shaker culture is 4-6 days; preferably, it is 5 days.
  • Sequence identity The percent sequence identity was determined using a computer program based on a dynamic programming algorithm.
  • Preferred computer programs within the scope of the present invention include the BLAST (Basic Local Alignment Search Tool) search program designed to explore all available sequence databases, whether the query is protein or DNA. Version 2.0 of BLAST (Gapped BLAST) of this search tool is already publicly available on the Internet (currently http://www.ncbi.nlm.nih.gov/BLAST/). It uses a heuristic algorithm that searches local alignments rather than global alignments to detect relationships between sequences that only share separate regions. Scores assigned in BLAST searches have well-defined statistical interpretations. The program preferably runs with selectable parameters set to default values.
  • Transformation refers to the introduction of exogenous nucleic acid into a cell. In particular, it refers to the stable integration of a DNA molecule into the genome of an organism of interest.
  • Chimeric lysozyme variant refers to a polypeptide comprising domains from two or more polypeptides, eg, a binding domain from one polypeptide and a catalytic domain from another polypeptide. Domains can be fused at the N-terminus or the C-terminus.
  • Lysozyme positive transformant specifically refers to the recombinant expression strain formed after the expression plasmid containing the lysozyme sequence is transformed into the host strain. As mentioned in Example 2 of the present invention, the lysozyme lyzAth-amdS expression plasmid is transformed into the host black. Recombinant expression strains formed in Aspergillus.
  • Lysozyme activity means 1, between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan or between N-acetyl-D-glucosamine residues in chitodextrin Hydrolysis of the 4-beta-bond, leading to lysis.
  • Lysozyme belongs to the enzyme class EC3.2.1.17. Lysozyme activity is typically measured by turbidimetric assays, eg, the turbidity change of a suspension of M. luteus CICC10680 induced by lysis of lysozyme. Under appropriate experimental conditions, these changes are proportional to the amount of lysozyme in the medium (c.f. INS 1105 of the Comprehensive Compendium of Food and Agriculture Organization Joint Food Additive Specifications (www.fao.org)).
  • Thermotolerance refers to elevated temperatures relative to the parental or identified reference sequence, in buffers or under conditions such as those present during product storage/transportation or similar to the use of the variant in industry Lysozyme activity after an incubation period under those conditions present in the process.
  • Animal Feed means any compound, formulation or mixture suitable or intended for ingestion by animals.
  • Animal feeds for monogastric animals typically include concentrates along with vitamins, minerals, enzymes, directly reared microorganisms, amino acids and/or other feed ingredients (eg in premixes), while animal feeds for ruminants typically include forages (including whole grains and silage), and may further include concentrates along with vitamins, minerals, enzymes, directly raised microorganisms, amino acids and/or other feed ingredients (eg, in premixes).
  • the beneficial effects of the present invention are as follows: the present invention constructs a chimeric novel lysozyme variant sequence and realizes its high-efficiency expression in Aspergillus niger, and its heat resistance is obviously improved compared with the parent.
  • a new lysozyme product can be developed, which can bring a new solution for protecting the intestinal health of animals and improving the utilization rate of feed under the background of "reducing and replacing resistance" in the national feed industry.
  • the solution can be applied commercially.
  • Figure 1 is a map of the lysozyme lyzAth expression plasmid.
  • Figure 2 is a map of the lysozyme lyzTte expression plasmid.
  • Figure 3 is a map of the lysozyme lyzAT expression plasmid.
  • Figure 4 is a map of the lysozyme lyzTA expression plasmid.
  • Figure 5 shows the residual rate of the three lysozymes in a water bath for 3 min at 60 °C.
  • Embodiment 1 Construction of lysozyme expression plasmid, comprising the following parts:
  • the pUC57 plasmid is linearized by vector-F and vector-R primers;
  • the lysozyme lyzAth derived from Aspergillus thermomutatus (Aspergillus thermomutatus) (its nucleotide sequence is as shown in SEQ ID NO:3, and its amino acid sequence is as shown in SEQ ID NO:4);
  • Thielavia terrestris Derived from the lysozyme lyzTte of Thermothielavioides terrestris (the Thielavia terrestris) (its nucleotide sequence is as shown in SEQ ID NO:5, and its amino acid sequence is as shown in SEQ ID NO:6);
  • the chimeric lysozyme variant lyzTA (its nucleotide sequence is as shown in SEQ ID NO:19, the amino acid sequence as shown in SEQ ID NO:19) of the chimeric lysozyme variant lyzTA of the first 109 amino acids of the N-terminal amino acid sequence of lysozyme lyzTte (as shown in SEQ ID NO:6) ID NO: 20);
  • the primers amdS-F and amdS-R, gla-F and gla-R were respectively amplified by PCR to amplify the amdS gene with the recombination arm and the DNA fragment containing the gla promoter and terminator, through the Gibson Master Mix Kit ( E2611, New England Biolabs) recombined the above-mentioned linearized pUC57 plasmid, amdS gene and gla promoter and terminator DNA fragment to obtain pGla-amdS plasmid, and the sequence was confirmed by sequencing.
  • This plasmid can be used for insertion of the lysozyme gene after linearization through the AflII site.
  • the lyzAth expression plasmid of lysozyme was constructed as follows: the primers lyzAth-F and lyzAth-R were used to amplify the lyzAth gene with the recombination arm by PCR, and then the lyzAth gene was combined with the linearized lyzAth gene by Gibson Master Mix Kit (E2611, New England Biolabs). The pGla-amdS plasmid was recombined to obtain the plyzAth-amdS plasmid. The sequence was confirmed by sequencing. The constructed plasmid map is shown in Figure 1. This plasmid can be used for protoplast transformation after linearization through the ApaI site.
  • the lyzTte expression plasmid of lysozyme was constructed as follows: the primers lyzTte-F and lyzTte-R were used to amplify the lyzTte gene with the recombination arm by PCR, and then the lyzTte gene was combined with the linearized lyzTte gene by Gibson Master Mix Kit (E2611, New England Biolabs). The pGla-amdS plasmid was recombined to obtain the plyzTte-amdS plasmid. The sequence was confirmed by sequencing, and the constructed plasmid map was shown in Figure 2. This plasmid can be used for protoplast transformation after linearization through the ApaI site.
  • the lyzAT expression plasmid of lysozyme was constructed as follows: the primers lyzAth-F and lyzTte-R were used to amplify the lyzAT gene with the recombination arm by PCR, and then the lyzAT gene was combined with the linearized lyzAT gene by Gibson Master Mix Kit (E2611, New England Biolabs). The pGla-amdS plasmid was recombined to obtain the plyzAT-amdS plasmid. The sequence was confirmed by sequencing, and the constructed plasmid map was shown in Figure 3. This plasmid can be used for protoplast transformation after linearization through the ApaI site.
  • the lyzTA expression plasmid of lysozyme was constructed as follows: the primers lyzTte-F and lyzAth-R were used to amplify the lyzTA gene with the recombination arm by PCR, and then the lyzTA gene was combined with the linearized pGla by Gibson Master Mix Kit (E2611, New England Biolabs). The -amdS plasmid was recombined to obtain the plyzTA-amdS plasmid. The sequence was confirmed by sequencing, and the constructed plasmid map was shown in Figure 4. This plasmid can be used for protoplast transformation after linearization through the ApaI site.
  • the relevant primer sequences are as follows:
  • enzymatic hydrolysis solution % (Sigma) and lysozyme (Sigma) 0.2% enzymatic hydrolysis solution (pH 5.8) at 30°C and 65 rpm for 3 h; then place the enzymatic hydrolysis solution containing protoplasts on ice and use four layers of The filtrate was gently centrifuged at 3000rpm and 4°C for 10min, the supernatant was discarded, and the protoplasts attached to the tube wall were filtered with STC solution (1M D-Sorbitol, 50mM CaCl 2 , 10mM Tris, pH 7.5 ) was washed once, and finally the protoplasts were resuspended in an appropriate amount of STC solution.
  • STC solution 1M D-Sorbitol, 50mM CaCl 2 , 10mM Tris, pH 7.5
  • Protoplast transformation Add 10 ⁇ l (concentration of 1000 ng/ ⁇ l) of the lysozyme expression plasmid obtained by linearization with ApaI to 100 ⁇ l of the protoplast suspension, mix well, place at room temperature for 25 minutes, and then add 900 ⁇ l of PEG solution in 3 times After mixing, place at room temperature for 25 minutes, then centrifuge at room temperature for 10 minutes at 3000 rpm, discard the supernatant, resuspend the protoplasts attached to the tube wall in 1 ml of STC solution, and incubate with acetamide that has been pre-cooled to about 45 °C Base (sucrose 3%, KCl 0.05%, K 2 HPO 4 ⁇ 3H 2 O 0.1%, FeSO 4 0.001%, MgSO 4 0.0244%, acetamide 0.06%, CsCl 0.34%) mixed and spread on a plate, and placed on the plate after solidification. Incubate in a 34°C incubator for 4-5 days
  • the above four lysozyme-positive transformants were inoculated into shake flasks containing 50ml of YPM medium (0.2% yeast extract, 0.2% peptone, and 2% maltose), and cultured on a shaker with a temperature of 34°C and a rotating speed of 220rpm for 5%. On the next day, the supernatant of the fermentation broth was collected by centrifugation, and the enzyme activity of lysozyme was determined.
  • YPM medium 0.2% yeast extract, 0.2% peptone, and 2% maltose
  • Lysozyme can hydrolyze the cell wall of bacteria, resulting in the dissolution of the cell wall of M. luteus and the decrease of the absorbance value of the solution.
  • One unit of lysozyme activity is defined as the amount of lysozyme required to cause an absorbance change of 0.001 per minute at 450 nm using a suspension of Micrococcus luteus at pH 6.2 at 25°C.
  • Micrococcus luteus CICC10680 (purchased from China Industrial Microorganism Collection and Management Center CICC).
  • Lysozyme standard egg white lysozyme.
  • Substrate solution Prepare 50 mL of M. luteus suspension with phosphate buffer. Substrates were incubated at 37°C for 30 min before use.
  • the substrate solution is stable for 2h at room temperature. Adjust the zero point of the spectrophotometer with phosphate buffer, then measure the absorbance of the substrate solution, the reading at 450nm should be 0.70 ⁇ 0.1.
  • a 1 the absorbance of the sample at 450 nm for 1 min;
  • a 2 The absorbance of the sample at 450 nm for 3 minutes;
  • m the mass of lysozyme in the sample preparation solution for analysis, in milligrams (mg);
  • the lysozyme lyzAth derived from Aspergillus thermomutatus and the lysozyme lyzTte derived from Thermothielavioides terrestris were treated in a water bath at 60°C for 3 minutes, and the residual rates of thermostable enzyme activity were 8% and 26%, respectively.
  • the constructed hybrid lysozyme The heat-resistant enzyme activity residual rate of the enzyme lyzAT under the same conditions reached 60%, and the heat resistance was greatly improved compared with the parental sequence, while the heat-resistant enzyme activity residue of the constructed hybrid lyzTA lyzTA under the same conditions was only 25%.
  • the parental lysozyme lyzTte is close to heat tolerance.
  • Test enzyme lysozyme lyzAT (500,000 U/g, detected by national standard method), and its amino acid sequence is shown in SEQ ID NO: 8.
  • a total of 700 1-day-old healthy Aiba Yijia (AA) male broilers were selected and randomly divided into 5 treatment groups. There was no significant difference in the average weight of each treatment group. There were 14 replicates in each treatment group, with 10 chickens per replicate. The 5 treatment groups were divided into 1 control group and 4 test groups. The amount of lyzAT added in the control group and each test group were 0, 250g/t, 500g/t, 1000g/t and 3000g/t, respectively.
  • the trial period was 42 days in total and was divided into two phases: early and late.
  • corn-soybean meal type basal diet was selected, referring to NRC (1994) chicken nutritional needs, Chinese chicken feeding standard (2004), Chinese feed composition and nutritional value table (2020), according to 0-21 days of age, 22-42 days of age
  • the diets of 2 stages of age were formulated, and the composition and nutritional levels of the basal diets are shown in Table 3.
  • the premix is provided per kilogram of diet: VD 3 2 750IU, VE 20IU, VK 3 2mg, VB 1 1.5mg, VB 2 6mg, Pantothenic acid 12mg, Niacin 20mg, VB 6 2.5mg, VB 12 2.03mg, Mn 75mg, Zn 75mg, Fe 95mg, Cu 10mg, I 0.6mg, Se 0.3mg.
  • the metabolizable energy is the calculated value, and the rest are the measured value.
  • the experiment was carried out in the experimental chicken farm of Shenyang Agricultural University, and the surrounding environment, chicken house and utensils were disinfected before the experiment.
  • the test chickens were reared in three-dimensional cages, and the 14 replicates of each group were arranged separately according to the different positions of the chicken cages, so that the test conditions of each group were the same except for the different lysozyme products and doses.
  • Free access to food and water, free ventilation, all-day light, feeding management and immunization were carried out in accordance with normal procedures, and data collection and recording were done on time.
  • the feed intake and consumption were recorded every day, and the broilers were weighed on an empty stomach on the 1st, 21st, and 42d of the test, and the average daily feed intake (ADFI), average daily feed intake (ADFI), average daily feed intake (ADFI), average daily feed intake (ADFI) and Daily weight gain (ADG) and feed to weight ratio (F/G). Carefully observe and record the death situation every day, and calculate the death rate.
  • ADFI Average daily feed intake
  • Average daily weight gain (ADG) weight gain of each treatment during the test period/(test days ⁇ number of chickens in each treatment);
  • Feed-to-weight ratio (F/G) average daily feed intake/average daily weight gain
  • Dead panning rate (%) number of dead panning chickens at the end of the test/number of broilers at the beginning of the test ⁇ 100%.
  • the European index is a comprehensive evaluation of the body weight, survival rate, feed-to-weight ratio, production management and other indicators of the flock, and reflects the level of profitability. The larger the index, the more profit.
  • the European Index is calculated as follows:
  • the comprehensive economic benefit refers to the sales price of broilers minus the production cost of broilers.
  • the main factors affecting the production cost of broilers are the feed-to-meat ratio, average feed price and chick cost.
  • the economic impact is huge.
  • EXCEL software was used for data analysis and processing
  • SPSS17.0 software was used for one-way ANOVA
  • Dun-can's method was used for multiple comparisons when the difference was significant, with P ⁇ 0.05 as the significant level.
  • P ⁇ 0.01 was extremely significant, and the results were expressed as "mean ⁇ standard deviation”.
  • the lysozyme lyzAT 250g/t test group was extremely significantly higher than the control group (P ⁇ 0.01), and the lysozyme lyzAT 500g/t test group was significantly higher than the control group (P ⁇ 0.01).
  • the rest of the experimental groups and the control group were not significantly different (P>0.05); in terms of average daily weight gain, the lysozyme lyzAT 250g/t and 500g/t test groups were extremely significantly higher than the control group (P ⁇ 0.01).
  • the 1000g/t test group was significantly higher than the control group (P ⁇ 0.05), and the 3000g/t test group was not significantly different from the control group (P>0.05).
  • the lysozyme lyzAT 250g/t, 500g/t and 1000g/t test groups were significantly higher than the control group (P ⁇ 0.01); , the lysozyme lyzAT 250g/t test group was significantly lower than the control group (P ⁇ 0.01), and the lysozyme lyzAT 500g/t test group was significantly lower than the control group (P ⁇ 0.05). The difference was not significant (P>0.05).
  • the price of chicks is 1.95 yuan/piece, the price of feather chickens is 8.4 yuan/kg, and the feed price is 3500 yuan/t.
  • Table 6 the average profit per chicken of each test group of lysozyme lyzAT is higher than that of the control group, and the average profit per chicken of the lysozyme lyzAT 250g/t test group is the highest, and the average profit per chicken is increased by 1.08 yuan than the control group.
  • the feed supplemented with lyzAT lyzAT can significantly increase the body weight, average daily gain and average daily feed intake of broilers at each stage, while significantly reducing the feed-to-weight ratio, European index and economic efficiency index.
  • the significant improvement of lyzAT also shows that the breeding benefit of lysozyme lyzAT is very considerable.

Abstract

提供了一种耐热性提升的嵌合溶菌酶变体及编码该变体的多核苷酸，同时构建一种新的嵌合溶菌酶序列，并实现在宿主细胞中的高效表达，该嵌合溶菌酶在60℃条件下的热稳定性较亲本序列提高了近一倍。还提供了一种嵌合溶菌酶变体的用途。还提供了一种含有溶菌酶变体的动物饲料添加剂及含有该动物饲料添加剂的动物饲料，所述饲料添加剂对动物生长性能具有显著的效果，大大提升了饲料的转化率。

Description

一种嵌合溶菌酶变体及其在动物饲料添加剂中的应用 技术领域
本发明属于生物工程技术领域，涉及一种嵌合溶菌酶变体及其在动物饲料添加剂中的应用。
背景技术
溶菌酶(Lysozyme,EC 3.2.1.17)是一种作用于微生物细胞壁的水解酶，又称为胞壁质酶。它能有效地水解细菌细胞壁的肽聚糖(peptidoglycan)，其机制主要是透过水解N-乙酰胞壁酸(N-acetylmuramic acid)和N-乙酰葡萄糖胺(N-acetylglucosamine)之间的β-1,4糖苷键，使肽聚糖骨架结构断裂后造成细胞壁破裂，最终导致细菌溶解。
溶菌酶天然存在于许多生物中，例如病毒、植物、昆虫、鸟类、爬行动物以及哺乳动物。在哺乳动物中，已经从鼻腔分泌物、唾液、眼泪、肠内容物、尿和乳中分离出溶菌酶。目前已经将溶菌酶分类为七种不同的糖苷水解酶家族(CAZy www.cazy.org)：GH18、GH19、鸡蛋清溶菌酶(GH22)、鹅蛋清溶菌酶(GH23)、噬菌体T4溶菌酶(GH24)、鞘氨醇单胞菌(Sphingomonas)鞭毛蛋白(GH73)以及拟鞘孢(Chalaropsis)溶菌酶(GH25)。溶菌酶是一种无毒、且对人和哺乳动物无副作用的蛋白质，因其具有溶菌的特性，近年来也已被广泛地应用于不同工业上。它在乳制品工业中可当作天然防腐剂，例如在巴氏杀菌奶添加溶菌酶能有效地延长其保存期。在食品应用上，可延长水产品及肉食品的储存时间。而在动物饲料工业中抗生素的长期、大量使用存在着严重的负面效应，畜禽耐药性情况日趋严重，产生大量高抗药性的菌株甚至新的致病菌株，不仅使动物疫病防控越来越困难，还严重影响了畜禽产品的质量及其安全性，直接影响到了人类的生命健康。国际社会已认识到抗生素的危害，许多国家已开始禁止在畜禽饲料中添加抗生素。在此背景下，溶菌酶作为一种非特异性免疫因子，在替抗方面具有重要的应用前景。溶菌酶可以催化水解细菌细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡糖之间的β-1,4糖苷键，导致细菌内容物渗出而使细菌溶解，从而实现抑菌等效果。同时，其可有效地分解受伤组织的脓液，增强防卫功能，从而有效保护消化道内膜，加快肠道和损伤组织的修复。另外，溶菌酶与聚合磷酸盐和甘氨酸等配合使用，具有良好的防腐作用，在饲料中添加溶菌酶可防止霉变，延长饲料的贮存期，减少不必要的损耗。溶菌酶与葡萄糖氧化酶配合使用还具有增效作用。
目前，溶菌酶在饲料及养殖生产中的应用有很大限制。主要有以下原因：一是目前绝大部分的溶菌酶几乎都是从鸡蛋清中提取，生产效率及产量较低，使用成本较高；二是大部分溶菌酶热稳定性较差，无法满足饲料高温造粒的工艺需求。因此，研究开发利用微生物发酵 法获得的低成本、热稳定性提升的新型溶菌酶产品具有重要的意义。
发明内容
为了解决上述技术的不足，本发明的一个目的是提供一种耐热性提升的嵌合溶菌酶变体及编码该变体的多核苷酸，构建了一种新的嵌合溶菌酶变体序列，并实现了其在宿主细胞中的高效表达，该嵌合溶菌酶变体在60℃条件下的热稳定性较亲本序列提高了近一倍；本发的另一个目的是提供一种耐热性提升的嵌合溶菌酶变体在动物饲料添加剂中的应用，所述饲料添加剂对动物生长性能具有显著的效果，大大提升了饲料的转化率。
本发明提供了一种嵌合溶菌酶变体，所述嵌合溶菌酶变体的氨基酸序列如SEQ ID NO:8所示，或与SEQ ID NO:8具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％以上的序列一致性；且该嵌合溶菌酶变体具有溶菌酶活性。
在一个优选方案中，所述的氨基酸序列如SEQ ID NO:8所示，即所述的氨基酸序列是将如SEQ ID NO:6所示的氨基酸序列的N端前109个氨基酸替换成如SEQ ID NO:4所示的氨基酸序列的N端前111个氨基酸得到的。
本发明还提供了所述的嵌合溶菌酶变体在制备替代抗生素类产品中的应用。其中，所述“替代抗生素类产品”是指可替代抗生素使用的产品，其可以用于动物饲料添加剂中，作为抗生素的潜在替代物，起到抑制有害微生物繁殖、保护动物肠道健康、提高动物免疫力等作用。
本发明还提供了如上所述的嵌合溶菌酶变体的用途，该用途：
在动物饲料中；
在动物饲料添加剂中；
在用于在动物饲料中使用的组合物的制备中；
用于改善动物饲料的营养价值；
用于提高动物饲料的消化率；和/或，
用于改善动物的一个或多个性能参数。
本发明还提供了一种用于改善动物饲料的营养价值的方法，向该饲料中添加如上所述的嵌合溶菌酶变体。
本发明还提供了一种动物饲料添加剂，其包含如上所述的嵌合溶菌酶变体。
进一步地，所述饲料添加剂进一步包含选自下组的一种或多种组分：
一种或多种维生素；
一种或多种矿物质；
一种或多种氨基酸；
一种或多种植生素；
一种或多种益生元；
一种或多种有机酸；以及
一种或多种其他饲料成分。
所述维生素包括脂溶性维生素和水溶性维生素；脂溶性维生素的非限制性实例包括维生素A、维生素D3、维生素E和维生素K，例如维生素K3；水溶性维生素的非限制性实例包括维生素C、维生素B12、生物素和胆碱、维生素B1、维生素B2、维生素B6、烟酸、叶酸和泛酸盐，例如Ca-D-泛酸盐。
所述矿物质的非限制性实例包括钙、镁、磷、钾、钠、硼、钴、氯化物、铬、铜、氟化物、碘、铁、锰、钼、硒和锌。
所述氨基酸的非限制性实例是赖氨酸、丙氨酸、β-丙氨酸、苏氨酸、甲硫氨酸和色氨酸。
所述植生素是一组来自草药、香料或其他植物的用作饲料添加剂的天然生长促进剂或非抗生素生长促进剂。植生素可以是由精油/提取物、单一植物和植物混合物(草药产品)或精油/提取物/植物的混合物(专门产品)制备的单一物质。植生素的实例是迷迭香、鼠尾草、牛至、百里香、丁香和柠檬草。精油的实例是百里酚、丁香酚、间甲酚、香草醛、水杨酸酯、间苯二酚、邻甲氧基苯酚(guajacol)、姜酚、薰衣草油、紫罗酮、鸢尾酮、桉叶油素、薄荷醇、薄荷油、α-蒎烯、柠檬烯、茴香脑、芳樟醇、二氢茉莉酸甲酯、香芹酚、丙酸/丙酸酯、乙酸/乙酸酯、丁酸/丁酸酯、迷迭香油、丁香油、香叶醇、萜品醇、香茅醇、水杨酸戊酯和/或水杨酸苄酯、肉桂醛、植物多酚(单宁)、姜黄和姜黄提取物。
所述益生元是诱导微生物(例如，细菌和真菌)生长或活动的物质，其有助于宿主的健康。益生元通常是不可消化的纤维化合物，其未消化地通过胃肠道的上部并刺激通过充当它们的底物而定殖大肠的有利细菌的生长或活动。通常，益生元增加胃肠(GI)道中双歧杆菌和乳酸菌的数量或活动。酵母衍生物(灭活的整个酵母或酵母细胞壁)也可以被认为是益生元。它们通常包括甘露寡糖、酵母β-葡聚糖或蛋白质内容物，并且通常衍生自酵母(酿酒酵母)的细胞壁。
所述有机酸在自然界中作为植物或动物组织的正常成分广泛分布。它们也通过主要在大肠中的碳水化合物的微生物发酵形成。它们通常在猪和家禽生产中用作抗生素生长促进剂的替代品，因为它们对鸡的坏死性肠炎和仔猪的大肠杆菌感染等肠道问题具有预防作用。有机酸可以作为单组分或通常2或3种不同有机酸的混合物出售。有机酸的实例是丙酸、甲酸、柠檬酸、乳酸、山梨酸、苹果酸、乙酸、富马酸、苯甲酸、丁酸和酒石酸或其盐(通常是钠或 钾盐、如二甲酸钾或丁酸钠)。
本发明的饲料添加剂可以进一步包含着色剂、稳定剂、生长改善添加剂和芳香化合物/调味品、多不饱和脂肪酸(PUFA)、活性氧产生物质、抗氧化剂、抗微生物肽、抗真菌多肽以及霉菌毒素控制化合物。
在一个优选方案中，所述嵌合溶菌酶变体的添加量为每吨动物饲料中添加嵌合溶菌酶变体100-1000g(100-1000g/t)；优选地，添加量为每吨动物饲料中添加嵌合溶菌酶变体250-500g(250-500g/t)。
本发明还提出了一种含有如上所述的动物饲料添加剂的动物饲料。
进一步地，所述饲料还包含基础饲粮。
所述基础饲粮参照NRC(1994)鸡营养需要、中国鸡饲养标准(2004)、中国饲料成分及营养价值表(2020)，按不同生长阶段分别配制。
在一个优选方案中，所述动物饲料添加剂的添加量为每吨动物饲料中添加动物饲料添加剂250-500g(250-500g/t)。
溶菌酶本身是一种天然蛋白质，无毒性，是一种安全性高的饲料添加剂。在不用任何抗生素的情况下，在饲料中添加饲用溶菌酶制剂，可促进动物对饲料中营养物质的消化吸收，提高动物增质量和饲料转化率，降低动物发病率和死亡率。
本发明还提供了一种多核苷酸，其编码如上任一项所述的嵌合溶菌酶变体。
在一个优选方案中，所述的多核苷酸的核苷酸序列如SEQ ID NO:7所示。
本发明还提供了含有如上所述的编码嵌合溶菌酶变体的多核苷酸的重组载体。
本发明还提供了含有如上所述的重组载体的宿主细胞。
其中，所述宿主细胞可以为黑曲霉、毕赤酵母、米曲霉、里氏木霉、枯草芽孢杆菌、地衣芽孢杆菌或大肠杆菌中的任意一种，本发明对宿主细胞的种类没有具体限制，利用常规实验方法可以成功表达本发明的嵌合溶菌酶变体即可。
本发明还提供了一种溶菌酶黑曲霉重组表达菌株的摇瓶培养方法，将溶菌酶阳性转化子接种至含有YPM培养基的摇瓶中，摇床培养，离心收集发酵液上清，并测定溶菌酶酶活。
其中，所述YPM培养基的组成及含量为：酵母提取物0.2％，蛋白胨0.2％，麦芽糖2％。
其中，所述摇床培养的温度为30-35℃；优选地，为34℃。
其中，所述摇床培养的转速为180-250rpm；优选地，为220rpm。
其中，所述摇床培养的时间为4-6天；优选地，为5天。
以下术语具体指：
序列一致性：序列一致性的百分比是用基于动态规划算法的计算机程序来确定的。在本 发明范围内优选的计算机程序包括设计用于探索所有可利用的序列数据库的BLAST(基础局部比对搜索工具(BasicLocal Alignment Search Tool))搜索程序，不管该查询是蛋白质还是DNA。本搜索工具的BLAST 2.0版(Gapped BLAST)已经是在因特网上公众可用的了(目前为http：//www.ncbi.nlm.nih.gov/BLAST/)。它使用了探索式算法，搜索局部比对而不是全局比对从而能够探测到仅共享分离的区域的序列间的关系。在BLAST搜索中指定的分值具有定义明确的统计学解释。该程序优选地将可选择的参数设为默认值来运行。
转化：指将外源核酸引入细胞。特别的，它指将DNA分子稳定地整合入目的生物体的基因组中。
嵌合溶菌酶变体：指包含来自两个或更多个多肽的结构域的多肽，例如，来自一个多肽的结合结构域和来自另一个多肽的催化结构域。结构域可以在N末端或C末端融合。
溶菌酶阳性转化子：具体指将包含溶菌酶序列的表达质粒转化到宿主菌株后形成的重组表达菌株，如在本发明实施例2中提到的将溶菌酶lyzAth-amdS表达质粒转化到宿主黑曲霉中形成的重组表达菌株。
溶菌酶活性：意指肽聚糖中N-乙酰胞壁酸和N-乙酰-D-葡糖胺残基之间或壳糊精中N-乙酰-D-葡糖胺残基之间的1,4-β-键的水解，导致溶菌作用。溶菌酶属于EC3.2.1.17酶类。溶菌酶活性通常通过比浊法测定来测量，例如由溶菌酶的裂解作用诱导的藤黄微球菌CICC10680的悬浮液的浊度变化。在适当的实验条件下，这些变化与培养基中溶菌酶的量成比例(c.f.联合国粮食及农业组织联合食品添加剂规格综合纲要的INS 1105(www.fao.org))。
耐热性：是指相对于亲本或鉴别的参考序列在升高的温度下，在缓冲液中或在例如在产品储存/运输过程中存在的那些条件下或类似于在工业使用该变体的过程中存在的那些条件下，在一段孵育期后的溶菌酶活性。
动物饲料：是指适合于或打算用于由动物摄入的任何化合物、制剂或混合物。单胃动物的动物饲料通常包括浓缩物连同维生素、矿物质、酶、直接饲养的微生物、氨基酸和/或其他饲料成分(例如在预混物中)，而反刍动物的动物饲料通常包括草料(包括粗粮和青贮)，并且可以进一步包括浓缩物连同维生素、矿物质、酶、直接饲养的微生物、氨基酸和/或其他饲料成分(例如在预混物中)。
本发明的有益效果在于：本发明构建了一种嵌合的新型溶菌酶变体序列并实现了其在黑曲霉中的高效表达，其耐热性较亲本都有明显的提高。此外，基于本发明可以开发出一种新的溶菌酶产品，该产品能够在国家饲料行业“减抗替抗”的大背景下带来一种保护动物肠道健康、提高饲料利用率的全新解决方案，可商业应用。
附图说明
图1是溶菌酶lyzAth表达质粒图谱。
图2是溶菌酶lyzTte表达质粒图谱。
图3是溶菌酶lyzAT表达质粒图谱。
图4是溶菌酶lyzTA表达质粒图谱。
图5是三种溶菌酶在60℃下水浴处理3min酶活残留率。
具体实施方式
结合以下具体实施例和附图，对发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1.溶菌酶表达质粒的构建，包含以下几个部分：
(1)将pUC57质粒通过vector-F与vector-R引物进行线性化；
(2)选择标记amdS表达盒，由GenScript公司合成，序列如SEQ ID NO:1所示；
(3)含有黑曲霉糖化酶基因gla启动子和终止子的DNA片段，由GenScript公司合成，序列如SEQ ID NO:2所示；
(4)溶菌酶基因序列：
来源于Aspergillus thermomutatus(温变曲霉)的溶菌酶lyzAth(其核苷酸序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示)；
来源于Thermothielavioides terrestris(太瑞斯梭孢壳霉)的溶菌酶lyzTte(其核苷酸序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示)；
将来源于Thermothielavioides terrestris溶菌酶lyzTte的氨基酸序列(如SEQ ID NO:6所示)的N端前109个氨基酸替换成来源于Aspergillus thermomutatus溶菌酶lyzAth的氨基酸序列(如SEQ ID NO:4所示)的N端前111个氨基酸，得到嵌合溶菌酶变体lyzAT(其核苷酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:8所示)；
将来源于Aspergillus thermomutatus(温变曲霉)的溶菌酶lyzAth的氨基酸序列(如SEQ ID NO:4所示)的N端前111个氨基酸替换成来源于Thermothielavioides terrestris(太瑞斯梭孢壳霉)的溶菌酶lyzTte的氨基酸序列(如SEQ ID NO:6所示)的N端前109个氨基酸的嵌合溶菌酶变体lyzTA(其核苷酸序列如SEQ ID NO:19所示，氨基酸序列如SEQ ID NO:20所示)；
上述基因序列由GenScript公司合成。
首先，分别将引物amdS-F与amdS-R、gla-F与gla-R通过PCR扩增出带有重组臂的amdS基因和含有gla启动子和终止子的DNA片段，通过Gibson Master Mix Kit(E2611，New England  Biolabs)将上述线性化pUC57质粒、amdS基因和gla启动子与终止子DNA片段进行重组得到pGla-amdS质粒，测序确认序列正确。该质粒可通过AflII位点线性化后用于溶菌酶基因的插入。
溶菌酶lyzAth表达质粒构建如下：将引物lyzAth-F与lyzAth-R通过PCR扩增出带有重组臂的lyzAth基因，再通过Gibson Master Mix Kit(E2611，New England Biolabs)将lyzAth基因与线性化的pGla-amdS质粒进行重组得到plyzAth-amdS质粒，经测序确认序列，构建好的质粒图谱见图1。该质粒可通过ApaI位点线性化后用于原生质体转化。
溶菌酶lyzTte表达质粒构建如下：将引物lyzTte-F与lyzTte-R通过PCR扩增出带有重组臂的lyzTte基因，再通过Gibson Master Mix Kit(E2611，New England Biolabs)将lyzTte基因与线性化的pGla-amdS质粒进行重组得到plyzTte-amdS质粒，经测序确认序列，构建好的质粒图谱见图2。该质粒可通过ApaI位点线性化后用于原生质体转化。
溶菌酶lyzAT表达质粒构建如下：将引物lyzAth-F与lyzTte-R通过PCR扩增出带有重组臂的lyzAT基因，再通过Gibson Master Mix Kit(E2611，New England Biolabs)将lyzAT基因与线性化的pGla-amdS质粒进行重组得到plyzAT-amdS质粒，经测序确认序列，构建好的质粒图谱见图3。该质粒可通过ApaI位点线性化后用于原生质体转化。
溶菌酶lyzTA表达质粒构建如下：将引物lyzTte-F与lyzAth-R通过PCR扩增出带有重组臂的lyzTA基因，再通过Gibson Master Mix Kit(E2611，New EnglandBiolabs)将lyzTA基因与线性化的pGla-amdS质粒进行重组得到plyzTA-amdS质粒，经测序确认序列，构建好的质粒图谱见图4。该质粒可通过ApaI位点线性化后用于原生质体转化。
相关引物序列如下：
表1本发明中的引物
Figure PCTCN2021130202-appb-000001
Figure PCTCN2021130202-appb-000002
实施例2.溶菌酶表达质粒plyzAth-amdS、plyzTte-amdS、plyzAT-amdS和plyzTA-amdS的转化整合
采用原生质体转化法分别将4种线性化溶菌酶表达质粒导入黑曲霉CICC2462菌株中(购自中国工业微生物菌种保藏管理中心CICC)，具体操作步骤如下：
(1)原生质体的制备：在营养丰富的TZ液体培养基(牛肉膏粉0.8％、酵母浸膏0.2％、蛋白胨0.5％、NaCl 0.2％、蔗糖3％，pH 5.8)中接种黑曲霉菌丝体，培养48h后采用Mira-cloth(Calbiochem公司)过滤收集菌丝体并用0.7M NaCl(pH 5.8)洗涤；待菌丝体滤干后转移至含纤维素酶1％(Sigma)、蜗牛酶1％(Sigma)和溶壁酶(Sigma)0.2％的酶解液(pH 5.8)中，在30℃、65rpm条件下酶解3h；然后将含有原生质体的酶解液置于冰上并用四层擦镜纸过滤，得到的滤液经3000rpm、4℃温和离心10min后，弃上清液，将附着在管壁上的原生质体用STC溶液(1M D-Sorbitol、50mM CaCl 2、10mM Tris，pH 7.5)洗涤一次，最后把原生质体重悬于适量的STC溶液中。
(2)原生质体转化：将10μl(浓度为1000ng/μl)用ApaI线性化得到的溶菌酶表达质粒加入到100μl原生质体悬浮液中混匀后室温放置25min，然后分3次共加入900μl PEG溶液，混匀后室温放置25min，然后在室温离心10min、转速为3000rpm，弃上清液，将附着于管壁上的原生质体重悬于1ml STC溶液中，与预先降温至45℃左右的乙酰胺培养基(蔗糖3％、KCl 0.05％、K 2HPO 4·3H 2O 0.1％、FeSO 40.001％、MgSO 40.0244％、乙酰胺0.06％、CsCl 0.34％)混合并铺平板，待平板凝固后放入34℃培养箱中培养4-5天，将转化子挑至新的乙酰胺培养基平板中，放入34℃培养箱中再培养4-5天，长出的转化子称为阳性转化子。
采用上述原生质体转化方法，将4种线性化溶菌酶表达质粒lyzAth-amdS、lyzTte-amdS、lyzAT-amdS和lyzTA-amdS分别转化到黑曲霉菌株中，得到4种溶菌酶阳性转化子。
实施例3.溶菌酶黑曲霉重组表达菌株的摇瓶培养
将上述四种溶菌酶阳性转化子分别接种至含有50mlYPM培养基(酵母提取物0.2％，蛋白胨0.2％，麦芽糖2％)的摇瓶中，在温度为34℃、转速为220rpm的摇床上培养5天，离心收集发酵液上清液，并测定溶菌酶酶活。
实施例4.溶菌酶活力的测定
溶菌酶活力的测定参考国标GB/T 1886.257-2016进行检测。
溶菌酶可水解细菌的细胞壁,造成藤黄微球菌细胞壁的溶解而引起溶液吸光度值的降低。
一个溶菌酶活力单位定义为25℃、pH 6.2条件下，使用藤黄微球菌悬浊液在450nm处每 分钟引起吸光度变化值为0.001所需溶菌酶的量。
试剂和材料
藤黄微球菌:CICC10680(购自中国工业微生物菌种保藏管理中心CICC)。
0.1mol/L磷酸盐缓冲液(pH 6.2)。
称取11.70g磷酸二氢钠(NaH 2PO 4·2H 2O)、7.86g磷酸氢二钠(Na 2HPO 4·12H 2O)及0.372g乙二胺四乙酸二钠(EDTA-2Na)于无菌水中并稀释定容至1000mL。调整缓冲溶液pH至6.2±0.1。
溶菌酶标准品:蛋清溶菌酶。
底物溶液:用磷酸盐缓冲液制备50mL藤黄微球菌悬浊液。使用前，底物于37℃培养30min。
该底物溶液室温下可稳定2h。以磷酸盐缓冲液调分光光度计零点，然后测定底物溶液的吸光度，450nm下读数应为0.70±0.1。
标准溶液的制备：
精确称取50mg蛋清溶菌酶标准品于50mL容量瓶中，用约25mL磷酸盐缓冲液搅拌溶解并稀释定容，充分混匀(如果需要，冷冻该溶液以备后续测定)。转移3mL上述标准制备溶液至100mL容量瓶中，用磷酸盐缓冲液搅拌溶解并稀释定容。
测定：
取3份标准溶液和3份试样溶液进行测定。25℃室温下，将1cm比色皿放入分光光度计，用磷酸盐缓冲液调整吸光度零点。吸2.9mL底物溶液于比色皿，最初450nm处吸光度应为0.70±0.10，3min之内初始吸光度值变化应小于或等于0.003时，方可开始测定。吸取0.1mL标准溶液加入底物溶液，充分混合。记录3min内吸光度值的变化，每15s记录一次吸光度值。每分钟吸光度值变化应在0.03～0.08，若不在要求范围需调整试样溶液的浓度。重复操作测定试样溶液。反应1min后稳定，计算时忽略最初1min的读数。
结果计算
酶活力X,按式(A.1)计算:
Figure PCTCN2021130202-appb-000003
式中:
A 1：试样在450nm处反应1min时的吸光度；
A 2：试样在450nm处反应3min时的吸光度；
m：用于分析的试样制备溶液中的溶菌酶质量，单位为毫克(mg)；
2：获得1min和3min吸光度读数所用的时间，单位为分钟(min)；
0.001：由单位溶菌酶每分钟引起的吸光度降低的值。
实施例5.溶菌酶耐热性的测定
将溶菌酶待测样品用磷酸盐缓冲液稀释至5000U/mL，在试管中加入4.5mL磷酸缓冲液，于不同温度下预热5min。取0.5mL稀释液至试管中，混匀，60℃下水浴处理3min，取出后冰水浴降至室温，按溶菌酶酶活检测方法检测酶活。计算：耐热酶活残留率(％)＝U X/U 0*100％，其中U 0为水浴处理前酶活，U X为水浴处理后酶活。检测结果如图4，来源于Aspergillus thermomutatus的溶菌酶lyzAth和来源于Thermothielavioides terrestris的溶菌酶lyzTte在60℃下水浴处理3min，耐热酶活残留率分别为8％和26％，构建的杂合溶菌酶lyzAT相同条件下的耐热酶活残留率达到了60％，耐热性较亲本序列有了大幅提升，而构建的杂合溶菌酶lyzTA相同条件下的耐热酶活残留只有25％，与亲本溶菌酶lyzTte耐热性接近。
表2溶菌酶的耐热酶活残留率检测
溶菌酶 U 0(U/mL) U X(U/mL) 耐热酶活残留率(％)
lyzAth 53710 4250 8
lyzTte 101310 26140 26
lyzAT 220000 131220 60
lyzTA 138120 35110 25
实施例6.
1、试验材料
试验动物：爱拔益佳(AA)雄性肉鸡。
试验用酶：溶菌酶lyzAT(50万U/g，国标法检测)，其氨基酸序列如SEQ ID NO:8所示。
2、试验设计
选取1日龄健康爱拔益佳(AA)雄性肉鸡700只，随机分为5个处理组，各处理组均重差异不显著。每个处理组设14个重复，每个重复10只鸡。将5个处理组分为1个对照组和4个试验组，对照组和各试验组的溶菌酶lyzAT添加量分别为：0、250g/t、500g/t、1000g/t和3000g/t。
试验期共42天，分为前期和后期2个阶段。
3、试验日粮及营养水平
本试验选用玉米—豆粕型基础饲粮，参照NRC(1994)鸡营养需要、中国鸡饲养标准(2004)、中国饲料成分及营养价值表(2020)，按0-21日龄、22-42日龄2个阶段饲粮进行配制，基础饲粮组成及营养水平见表3。
表3基础日粮配方及营养水平(风干基础)％
项目 0-21日龄 项目 22-42日龄
原料   原料  
玉米 56.76 玉米 57.92
去皮豆粕 26.5 去皮豆粕 26
玉米蛋白粉 3.2 玉米蛋白粉 3
细石粉 1.1 细石粉 1.1
磷酸氢钙 1.1 磷酸氢钙 1.1
鸡油 6.5 鸡油 6
谷氨酸渣 2 谷氨酸渣 2
羽毛粉 0.5 羽毛粉 0.5
氯化钠 0.28 氯化钠 0.28
赖氨酸70％ 0.78 赖氨酸70％ 0.77
蛋氨酸99％ 0.22 苏氨酸98.5％ 0.1
伴侣B酶 0.05 蛋氨酸99％ 0.2
葡萄糖氧化酶 0.01 伴侣B酶 0.02
预混1) 1 葡萄糖氧化酶 0.01
预混1) 1    
合计 100 合计 100
营养水平      
代谢能(MJ/kg)2) 11.42   13.49
粗蛋白质 20.0   20.5
粗脂肪 4.21   5.51
钙 0.97   0.77
磷 0.41   0.32
注，表3中：
1)预混料为每千克饲粮提供：VD 32 750IU，VE 20IU，VK 32mg，VB 11.5mg，VB 26mg，泛酸12mg，烟酸20mg，VB 62.5mg，VB 122.03mg，Mn 75mg，Zn 75mg，Fe 95mg，Cu 10mg，I 0.6mg，Se 0.3mg。
2)代谢能为计算值，其余为实测值。
4、饲养管理
试验在沈阳农业大学试验鸡场进行，试验前对周边环境、鸡舍、用具进行消毒处理。试验鸡采用立体笼式饲养方法，每组的14个重复按鸡笼的位置不同分开排列，使试验影响因素除溶菌酶产品和剂量不一样外各组的试验条件均保持一致。自由采食及饮水，自由通风，全天光照，饲养管理和免疫按照正常程序进行，按时做好数据采集和记录。
5、测定指标
5.1生产性能
饲养试验期间，每天记录投料量和耗料量，并在试验的第1d、21d和42d对每重复肉鸡进行空腹称重，计算各阶段及整个生长期的平均日采食量(ADFI)、平均日增重(ADG)和料重比(F/G)。每天仔细观察和记录死淘情况，计算死淘率。
平均日采食量、平均日增重、料重比、死淘率的计算方法如下：
平均日采食量(ADFI)＝试验期各处理耗料量/(试验天数×各处理鸡数)；
平均日增重(ADG)＝试验期各处理增重/(试验天数×各处理鸡数)；
料重比(F/G)＝平均日采食量/平均日增重；
死淘率(％)＝试验结束死淘鸡数/试验初期肉鸡数×100％。
5.2欧洲指数
所述欧洲指数是通过对鸡群的体重、成活率、料重比、生产管理等指标进行综合性评价，体现盈利的高低，指数越大表明盈利越多。
所述欧洲指数的计算方法如下：
欧洲指数＝成活率×体重(kg)/(料肉比×出栏天数)×10000
5.3经济效益指标计算
所述综合经济效益是指肉鸡的销售价格减去肉鸡的生产成本，其中影响肉鸡生产成本的主要因素是料肉比、平均料价和鸡苗成本，肉鸡出栏时的市场价格也对肉鸡饲养的经济效益影响很大。
根据耗料、生长情况等数据比较对照组与试验组的综合经济效益。
5.4数据处理及统计分析
试验数据记录与整理：EXCEL软件进行数据分析处理，SPSS17.0软件进行单因素方差分析(one-way ANOVA)，差异显著时采用Dun-can氏法进行多重比较，以P<0.05为显著水平，P<0.01极显著水平，结果用“平均数±标准差”表示。
结果与分析
1、溶菌酶lyzAT对肉鸡生产性能的影响
由表4可知，0-21日龄，在肉鸡末重方面，溶菌酶lyzAT 500g/t和1000g/t试验组显著高于对照组(P<0.05)，其余各试验组与对照组相比差异均不显著(P>0.05)；在肉鸡平均日增重方面，溶菌酶lyzAT 500g/t和1000g/t试验组显著高于对照组(P<0.05)，其余各试验组与对照组相比差异均不显著(P>0.05)；在料重比方面，各试验组较对照组有降低趋势(P＝0.089)，溶菌酶lyzAT 250g/t、500g/t和1000g/t试验组均降低了1.55％；平均日采食量各试验组较对照组差异均不显著(P>0.05)。
从22-42日龄来看，在肉鸡末重方面，溶菌酶lyzAT 250g/t试验组极显著高于对照组(P<0.01)，溶菌酶lyzAT 500g/t试验组显著高于对照组(P<0.05)，其余各试验组与对照组差异均不显著(P>0.05)；在平均日增重方面，溶菌酶lyzAT 250g/t和500g/t试验组极显著高于对照组(P<0.01)，1000g/t试验组显著高于对照组(P<0.05)，3000g/t试验组与对照组差异不显著(P>0.05)；在料重比方面，溶菌酶lyzAT 250g/t和500g/t试验组显著低于对照组(P<0.05)，其余各试验组与对照组差异均不显著(P>0.05)；在平均日采食量方面，各试验组较对照组有增加趋势(P＝0.075)，其中,溶菌酶lyzAT 500g/t试验组较对照组相比增加了2.50％。
从0-42日龄来看，在平均日增重方面，溶菌酶lyzAT 250g/t、500g/t和1000g/t试验组均极显著高于对照组(P<0.01)；在料重比方面，溶菌酶lyzAT 250g/t试验组极显著低于对照组(P<0.01)，溶菌酶lyzAT 500g/t试验组显著低于对照组(P<0.05)；在平均日采食量方面各试验组差异不显著(P>0.05)。
表4溶菌酶lyzAT对肉鸡生产性能的影响
Figure PCTCN2021130202-appb-000004
注：同行数据肩标小写字母不同表示差异显著(P<0.05)，大写字母表示差异极显著(P<0.01)，相同或无字母表示差异不显著(P>0.05)。
2、溶菌酶lyzAT对肉鸡欧洲指数的影响
由表5可知，对照组试验全期的欧洲指数较各试验组均为最低，溶菌酶lyzAT 250g/t试验组试验全期的欧洲指数最高，比对照组高62.58，说明添加溶菌酶可提高肉鸡盈利。
表5溶菌酶lyzAT对肉鸡欧洲指数的影响
Figure PCTCN2021130202-appb-000005
3、溶菌酶lyzAT对肉鸡养殖效益的影响
雏鸡价格1.95元/只，毛鸡价格8.4元/kg，饲料价格3500元/t。由表6可知，溶菌酶lyzAT各试验组平均每只鸡利润均高于对照组，其中溶菌酶lyzAT 250g/t试验组平均每只鸡利润最高，比对照组平均每只鸡增加1.08元利润。
表6溶菌酶lyzAT对肉鸡养殖效益的影响
Figure PCTCN2021130202-appb-000006
因此，从上述试验数据中可以看出，添加了溶菌酶lyzAT的饲料能够显著提高肉鸡各阶段体重、平均日增重和平均日采食量，同时显著降低料重比，欧洲指数和经济效益指标的显著提高也说明溶菌酶lyzAT的养殖效益非常可观，其添加量可以为100-1000g/t，最适宜的添加剂量为250-500g/t。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。

Claims (11)

  1. 一种嵌合溶菌酶变体，其特征在于，所述嵌合溶菌酶变体的氨基酸序列如SEQ ID NO:8所示，或与SEQ ID NO:8具有至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％以上的序列一致性；且具有溶菌酶活性。
  2. 如权利要求1所述的嵌合溶菌酶变体，其特征在于，所述嵌合溶菌酶变体的氨基酸序列如SEQ ID NO:8所示。
  3. 如权利要求1所述的嵌合溶菌酶变体在制备替代抗生素类产品中的应用。
  4. 一种多核苷酸，其特征在于，编码如权利要求1或2中任一项所述的嵌合溶菌酶变体。
  5. 如权利要求4所述的多核苷酸，其特征在于，所述多核苷酸的核苷酸序列如SEQ ID NO:7所示。
  6. 一种重组载体，其特征在于，含有如权利要求4或5中任一项所述的多核苷酸。
  7. 一种宿主细胞，其特征在于，含有如权利要求6所述的重组载体。
  8. 如权利要求7所述的宿主细胞，其特征在于，所述宿主细胞选自黑曲霉、毕赤酵母、米曲霉、里氏木霉、枯草芽孢杆菌、地衣芽孢杆菌或大肠杆菌中的任意一种。
  9. 如权利要求1所述的嵌合溶菌酶变体的用途，其特征在于，该用途：
    在动物饲料中；
    在动物饲料添加剂中；
    在用于在动物饲料中使用的组合物的制备中；
    用于改善动物饲料的营养价值；
    用于提高动物饲料的消化率；和/或
    用于改善动物的一个或多个性能参数。
  10. 一种动物饲料添加剂，其特征在于，其包含如权利要求1所述的嵌合溶菌酶变体。
  11. 如权利要求10所述的动物饲料添加剂，其特征在于，所述嵌合溶菌酶变体的添加量为每吨动物饲料中添加嵌合溶菌酶变体100-1000g；优选地，添加量为每吨动物饲料中添加嵌合溶菌酶变体250-500g。
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