Phage lyase composite powder and preparation method and application thereof

Abstract

The invention relates to phage lyase composite powder. The composite powder composition comprises the following components in percentage by mass: 0.15%-0.2% of phage lyase TSPpgh, 0.15%-0.2% of phagelyase MMPpgh, 0.3%-0.5% of trehalose, 0.1%-0.2% of citric acid, 0.2%-0.3% of mannitol, 0.2%-0.3% of vitamin C and 98.3%-98.9% of diatomite. The phage lyase composite powder is subjected to in-vitro antibacterial activity detection by adopting a two layer plating method. After the composite powder is stored for 12 months at normal temperature, the enzyme activity is kept at 95% or above (the enzymeactivity loss is within 5%). The composite lyase preparation can be used for replacing antibiotics to inhibit proliferation of harmful pathogenic bacteria such as escherichia coli, salmonella and staphylococcus aureus in animal intestines, and can maintain intestinal microecological balance.

Description

technical field

[0001] The invention belongs to the technical field of enzyme preparations, and in particular relates to a composite powder of phage lyase and its preparation method and application. Background technique

[0002] Phage lyase is a type of cell wall hydrolytic enzyme that can hydrolyze peptidoglycan. After phage infects the host, the double-stranded DNA phage destroys the bacterial cell wall structure through the holin-lyase cleavage system, thereby causing the host to rupture. Lyases generally have two to three domains involved in the catalysis and binding of substrates. As a new type of bactericidal agent, the high affinity of lyase is related to the species-specific cell wall sugar, and the latter is a necessary component for the survival of bacteria, so it is difficult for bacteria to develop resistance to lyase. In nature, almost all All bacteria have corresponding phages, so the development and research of lyase is of great value. There are two ways for...

Examples

Embodiment 1

[0022] Example 1: The composition and mass percentage of the phage lyase compound powder are phage lyase TSPpgh 0.15%, phage lyase MMPpgh 0.2%, trehalose 0.5%, citric acid 0.1%, mannitol 0.2%, vitamin C0.2 %, diatomaceous earth 98.65%.

[0023] The preparation method of the above-mentioned phage lyase compound powder is as follows: the phage lyase TSPpgh gene and the phage lyase MMPpgh gene were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into E. coli BL21 to obtain The genetically engineered bacteria with TSPpgh gene and the genetically engineered bacteria with phage lyase MMPpgh gene were inoculated into 300mL seed medium respectively, and cultivated to OD at 37°C 600 =2.0, and then inoculated into the fermentation medium (using 20L automatic fermenter (Shanghai Bailun Biotechnology Co., Ltd.)) according to the inoculum amount of 2%, and cultivated at 37°C and 200rmp / min for 10h to reach the biomass of genetically...

Embodiment 2

[0025] Example 2: The composition and mass percentage of the phage lyase compound powder in this example are phage lyase TSPpgh 0.2%, phage lyase MMPpgh 0.15%, trehalose 0.3%, citric acid 0.2%, mannitol 0.3%, vitamin C 0.3%, diatomaceous earth 98.55%.

[0026] The preparation method of the above-mentioned phage lyase compound powder is as follows: the phage lyase TSPpgh gene and the phage lyase MMPpgh gene were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into E. coli BL21 to obtain The genetically engineered bacteria with TSPpgh gene and the genetically engineered bacteria with phage lyase MMPpgh gene were inoculated into 300mL seed medium respectively, and cultivated to OD at 37°C 600 =1.5, then inoculated into the fermentation medium (using a 20L automatic fermenter (Shanghai Bailun Biotechnology Co., Ltd.)) at an inoculation amount of 1.5%, and cultivated at 37°C and 150rmp / min for 12h to reach the biomass of gen...

Embodiment 3

[0028] Example 3: In vitro antibacterial activity detection of phage lytic enzyme composite powder

[0029] 1. Take 1 g of the phage lytic enzyme composite powder prepared in the above examples in a 4 mL EP tube, and take 1 g of sterilized diatomaceous earth in a 4 mL EP tube as a control;

[0030] 2. Add 3 mL PBS to the experimental group and the control group respectively;

[0031] 3. Diluted to 10 in the experimental group and the control group respectively -5 200 μL of the bacteria solution to be tested;

[0032] 4. After fully shaking, incubate in a 37°C water bath for 30 minutes, shaking every 5 minutes;

[0033] 5. After incubation, take 300 μL of double-layer medium for detection, and incubate at 37°C for 12 hours for colony counting;

[0034] The components of the lower solid medium are: NaCl 10.0g / L, tryptone 10.0g / L, yeast powder 5.0 g / L, agar powder 15.0 g / L;

[0035] The upper semi-solid medium components are: NaCl 10.0g / L, tryptone 10.0g / L, yeast powder: 5.0 ...

technical field

[0001] The invention belongs to the technical field of enzyme preparations, and in particular relates to a composite powder of phage lyase and its preparation method and application. Background technique

[0002] Phage lyase is a type of cell wall hydrolytic enzyme that can hydrolyze peptidoglycan. After phage infects the host, the double-stranded DNA phage destroys the bacterial cell wall structure through the holin-lyase cleavage system, thereby causing the host to rupture. Lyases generally have two to three domains involved in the catalysis and binding of substrates. As a new type of bactericidal agent, the high affinity of lyase is related to the species-specific cell wall sugar, and the latter is a necessary component for the survival of bacteria, so it is difficult for bacteria to develop resistance to lyase. In nature, almost all All bacteria have corresponding phages, so the development and research of lyase is of great value. There are two ways for...

Examples

Embodiment 1

[0022] Example 1: The composition and mass percentage of the phage lyase compound powder are phage lyase TSPpgh 0.15%, phage lyase MMPpgh 0.2%, trehalose 0.5%, citric acid 0.1%, mannitol 0.2%, vitamin C0.2 %, diatomaceous earth 98.65%.

[0023] The preparation method of the above-mentioned phage lyase compound powder is as follows: the phage lyase TSPpgh gene and the phage lyase MMPpgh gene were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into E. coli BL21 to obtain The genetically engineered bacteria with TSPpgh gene and the genetically engineered bacteria with phage lyase MMPpgh gene were inoculated into 300mL seed medium respectively, and cultivated to OD at 37°C 600 =2.0, and then inoculated into the fermentation medium (using 20L automatic fermenter (Shanghai Bailun Biotechnology Co., Ltd.)) according to the inoculum amount of 2%, and cultivated at 37°C and 200rmp / min for 10h to reach the biomass of genetically...

Embodiment 2

[0025] Example 2: The composition and mass percentage of the phage lyase compound powder in this example are phage lyase TSPpgh 0.2%, phage lyase MMPpgh 0.15%, trehalose 0.3%, citric acid 0.2%, mannitol 0.3%, vitamin C 0.3%, diatomaceous earth 98.55%.

[0026] The preparation method of the above-mentioned phage lyase compound powder is as follows: the phage lyase TSPpgh gene and the phage lyase MMPpgh gene were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into E. coli BL21 to obtain The genetically engineered bacteria with TSPpgh gene and the genetically engineered bacteria with phage lyase MMPpgh gene were inoculated into 300mL seed medium respectively, and cultivated to OD at 37°C 600 =1.5, then inoculated into the fermentation medium (using a 20L automatic fermenter (Shanghai Bailun Biotechnology Co., Ltd.)) at an inoculation amount of 1.5%, and cultivated at 37°C and 150rmp / min for 12h to reach the biomass of gen...

Embodiment 3

[0028] Example 3: In vitro antibacterial activity detection of phage lytic enzyme composite powder

[0029] 1. Take 1 g of the phage lytic enzyme composite powder prepared in the above examples in a 4 mL EP tube, and take 1 g of sterilized diatomaceous earth in a 4 mL EP tube as a control;

[0030] 2. Add 3 mL PBS to the experimental group and the control group respectively;

[0031] 3. Diluted to 10 in the experimental group and the control group respectively -5 200 μL of the bacteria solution to be tested;

[0032] 4. After fully shaking, incubate in a 37°C water bath for 30 minutes, shaking every 5 minutes;

[0033] 5. After incubation, take 300 μL of double-layer medium for detection, and incubate at 37°C for 12 hours for colony counting;

[0034] The components of the lower solid medium are: NaCl 10.0g / L, tryptone 10.0g / L, yeast powder 5.0 g / L, agar powder 15.0 g / L;

[0035] The upper semi-solid medium components are: NaCl 10.0g / L, tryptone 10.0g / L, yeast powder: 5.0 ...