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Geobacillus sp and application thereof

A geobacillus and rod-shaped technology, applied in the field of microorganisms, can solve the problems of reduced enzyme activity, inability to directly use feathers, poor thermal stability of keratinase, etc., and achieve fast degradation, high temperature resistance, and good thermal stability Effect

Inactive Publication Date: 2017-05-10
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the known microorganisms producing keratinase belong to room temperature bacteria, and the enzyme reaction temperature is generally between 40-60°C. The produced keratinase has disadvantages such as poor thermal stability, and most microorganisms cannot directly use untreated feathers.
After He Zhoufeng ("Identification of Efficient Feather Degrading Strains and Optimization of Fermentation Conditions", He Zhoufeng et al., Genomics and Applied Biology, 2015) optimized the enzyme production conditions of Bacillus subtilis producing keratinase, the enzyme activity is 23.6U / ml, and the optimum temperature for enzymatic reaction is 37°C; Microbiology Bulletin, 2014) The half-life of the protease at 60°C before modification was 9 minutes. Liu Bohong et al. carried out segmental deletion of the first 5 amino acids at the N-terminus of keratinase, and replaced the first 5 amino acids at the N-terminus with those from Thermoactinomyces by sequence alignment. The N-terminus of the thermophilic protease of vulgaris, the wild-type and mutant keratinase genes were expressed in Bacillus subtilis WB600, the half-life at 60°C was increased to 20min, but the enzyme activity was reduced by 70%

Method used

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  • Geobacillus sp and application thereof
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  • Geobacillus sp and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 strain breeding

[0038] 1. Screen strains

[0039] Sampling from the high-temperature compost of livestock and poultry manure (Nanjing Ningliang Organic Fertilizer Factory), first take a certain sample from each sample, break up the sample with sterile saline; after gradient dilution, inoculate in the enrichment medium for 60- Enrichment and subculture at 80°C for 2-3 days; streak the plate to separate, obtain high-temperature bacteria, spot the high-temperature bacteria on the separation medium plate, screen the strains with transparent hydrolysis circles, and then inoculate these strains into the screening medium , observe the degradation of feathers, and finally screen out a strain that has the ability to degrade feathers, and inoculate the strain into the slant solid medium for use.

[0040] 2. Identification of strains

[0041] A) Streak the strains screened in step 1 on the beef extract peptone medium, cultivate at 60°C for 24h, and obtain a single...

Embodiment 2

[0046] Embodiment 2 strain JAAS-YTC01 fermentation enzyme production condition optimization

[0047] Adopt single factor test to investigate adding different carbon sources (1%) (W / V, g / l) and different nitrogen sources and the addition amount of different concentration feathers to the bacterial strain JAAS-YTC01 keratinase yield that embodiment 1 obtains.

[0048] a Screening of the most suitable carbon source

[0049] Add 1% (W / V, g / l) of glucose, sucrose, maltose, starch, bran, and corn flour to the fermentation basal medium, and measure the enzyme activity after 48 hours of fermentation at 60° C. and 180 rpm in a shaking flask. The results showed that the addition of sucrose could increase the production of keratinase.

[0050] b Screening of the most suitable nitrogen source

[0051] Add 1% (W / V) yeast powder, soybean meal, 0.4% (W / V) urea, and ammonium sulfate to the fermentation basal medium, and measure the enzyme activity after 48 hours of fermentation at 60° C. and...

Embodiment 3

[0055] Optimum reaction temperature, optimum pH and thermostability of embodiment 3 bacterial strain JAAS-YTC01 keratinases

[0056] The bacterial strain JAAS-YTC01 screened in Example 1 was inoculated into the fermentation medium with an inoculum size of 2% (W / V, g / l), after 180rpm stirring and culturing for 48h, it was centrifuged at 8000rpm for 10min, and the gained supernatant was the horn protease solution.

[0057] Take 1ml of keratinase solution diluted 5-10 times, add 1ml of 1% keratin, carry out enzyme reaction at 60°C for 15min, then add 2ml of 0.4M TCA to stop the enzyme activity, and centrifuge at 4000rpm for 10min. Add 2ml 0.4M TCA first and then add 1% substrate as blank control. Take 1ml supernatant, add 5ml 0.4M Na 2 CO 3 Solution, then add 1ml of Folin's phenol reagent, keep warm at 40°C for 20min, and measure the absorbance at 660nm.

[0058] Using keratin as a substrate, the amount of enzyme required to catalyze the decomposition of keratin to produce 1 ...

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Abstract

The invention discloses geobacillus sp JAAS-YTC01 and a method of generating keratinase. A strain of the geobacillus sp is preserved in China General Microbiological Culture Collection Center on July 6, 2016, and has a preservation number of CGMCC No.12740; the strain of the geobacillus sp disclosed by the invention can generate the keratinase, feathers can be completely degraded in 48 hours, the problem of recycling waste feathers can be effectively solved, available amino acid and polypeptide are obtained, environment pollution is reduced, and the generated keratinase has thermal stability and is suitable for industrial production utilization.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a Geobacillus sp. JAAS-YTC01 and its application in degrading feathers. Background technique [0002] Keratin is derived from ectoderm cells. It is a kind of hard protein that is insoluble in water, dilute acid or dilute alkali solution, has high mechanical strength and stable chemical properties. It is widely found in hair, hair, scales, feathers, nails, hooves, Horns, claws, beaks, silk and other epidermal structures. The total amount of feathers in my country exceeds 1 million tons every year, and with the rapid development of intensive farming, the output is increasing year by year. Up to 90% of feathers are keratin. Analysis of the nutritional components of feathers shows that the amino acid content is more than 70%, which is a potential excellent protein resource. However, the disulfide bond in the structure of keratin makes it resistant to degradation and is not ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/52C12P21/06C12R1/01
CPCC12N9/52C12P21/06C12N1/205C12R2001/01
Inventor 王琳黄红英钱玉婷常志州
Owner JIANGSU ACAD OF AGRI SCI