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Keratinase-producing streptomyces rutgersensis LT-2 and application method thereof

A technology of keratinase and LT-2, which is applied in the field of microorganisms and achieves the effects of wide degradation spectrum, mild degradation conditions and simple operation process

Active Publication Date: 2015-11-04
湖南省微生物研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disulfide bond in the structure of keratin makes it resistant to degradation and is not degraded by most proteases such as papain, pepsin and trypsin.

Method used

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  • Keratinase-producing streptomyces rutgersensis LT-2 and application method thereof
  • Keratinase-producing streptomyces rutgersensis LT-2 and application method thereof
  • Keratinase-producing streptomyces rutgersensis LT-2 and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Screening and identification of keratinase producing strains

[0022] 1. Medium formula

[0023] (1) Preliminary screening of solid medium: 10-15 grams of pig hair powder, K per 1000 ml 2 HPO 4 1.0~1.4g, KH 2 PO 4 0.5~1.0g, NaCl 0.1~0.5g, MgSO 4 0.05 to 0.1 grams, 15 to 20 grams of agar, the balance is water. pH 7.5~8.0.

[0024] The preparation of the screening medium pig hair powder: the pig hair is removed from the pig skin, washed, dried to a constant weight, and then cut into about 1 cm of the pig hair with scissors, then crushed with a grinder, and passed through a 100-mesh sieve.

[0025] (2) Inclined preservation medium

[0026] Gao's medium: contains 15-20 grams of soluble starch, KNO per 1000 ml 3 0.8~1.2g, NaCl0.5~1.0g, K 2 HPO 4 0.5~1.0g, MgSO 4 0.5~1.0g, FeSO 4 ·7H 2 O 0.005~0.01 g. pH 7.2~7.5, agar 15~20 g, the balance is water.

[0027] LB solid medium: 8-12 grams of protein per 1000ml, 3-7 grams of yeast powder, 8-12 grams of NaCl, pH 7.0-7.5, 15-2...

Embodiment 2

[0047] Example 2 Determination of the degradation effect of strains

[0048] 1. Determination of keratinase activity

[0049] Suspend 5mg of pig hair powder in 1ml of 50mmol / L Tris buffer (pH8.0); add 1ml of crude enzyme solution diluted 10 times with the same Tris buffer; add 2ml of TCA( 0.4mol / L) stop solution; the reaction is heated at 50℃, 150rpm for 45min; add 2ml of TCA (0.4mol / L) to stop the reaction; the reaction solution is centrifuged at 6000rpm for 10min; the supernatant is colorimetrically measured at the wavelength of 280nm by spectrophotometer OD value.

[0050] Enzyme activity unit definition: Under certain reaction conditions, the amount of enzyme produced per milliliter of decomposing keratin increases the OD value by 0.01, which is 1 U of keratinase activity unit.

[0051] The preparation of the enzyme reaction substrate pig hair powder: the method is the same as the preparation of the screening medium pig hair powder, except that the pig hair powder that has been c...

Embodiment 3

[0056] Example 3 Method for expanding culture of strain LT-2

[0057] 3. The expansion method of strain LT-2 is

[0058] Strain LT-2 was inoculated on the slope of the solid seed culture medium, activated and cultured at 30-35°C for 2 to 3 days, until a large number of spores formed on the slope.

[0059] Scrape the spores from the activated slant, make a uniform spore suspension with sterile water, and then inoculate the spore suspension into the sporulation medium, culture at 30-35℃, 200rpm shaker for 3-5 days, the resulting shake The bottle culture liquid is the seed liquid.

[0060] Fermentation tank culture: the culture medium in the fermentation tank is 50-70% of the total volume, the inoculum is 1-3% of the volume of the culture medium, the fermentation temperature is 30-35°C, sterile air is introduced, and the culture is stirred at 180-200rpm , The ratio of the aeration volume to the volume of the fermentation broth is 0.9:1 to 1:1, the tank pressure is 0.03-0.05Mpa; the end ...

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Abstract

The invention relates to a keratinase-producing streptomyces rutgersensis LT-2 and an application method thereof, and belongs to the technical field of microbes. The bacterial strain LT-2 has the classification name of streptomyces rutgersensis, and the preservation number is CCTCC NO: M 2015440. The invention discloses the morphological characteristics of the bacterium on GAUZE's medium No.1, and the morphological characteristics comprise that aerial mycelia is yellow-white, spores in a pile are yellow, substrate mycelium is light yellow-brown; sporogeneous mycelium is straight or wavy; and conidium is from sphere to ellipse, and the spore surface is smooth. LT-2 is applied to degrade alpha-keratin such as hairs of human and animals, hoofs, pawls, horns, shells and the like, and is applied to degrade beta-keratin such as feathers, bird bills, reptile toenails, squamas, claws and the like. Compared with other bacterial strains degrading keratins, the bacterial strain disclosed by the invention is mild in fermentation conditions and convenient to apply, and the produced keratinase is relatively high in enzyme activity and relatively stable.

Description

Technical field [0001] The invention belongs to the technical field of microorganisms, and relates to a novel strain of Streptomyces rutjesii LT-2 producing keratinase and an application method thereof. Background technique [0002] Keratin can be divided into two types: alpha keratin and beta keratin. Alpha keratin is mainly found in mammal hair, feathers, horns, nails, paws and hoofs. The harder beta keratin is found in the toenails, shells, scales, beaks, claws and bristles of reptiles. Its crude protein content is more than 80%, the total amount of various amino acids is more than 70%, and it contains more macro elements, trace elements and unknown growth factors. It is a good source of feed protein and fertilizer, and it is useful for its development and utilization. Important application prospects. [0003] In recent years, with the rapid development of large-scale breeding, a large amount of livestock and poultry wastes have been produced, mainly in the main forms of hair,...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/52C12R1/465
Inventor 杜东霞许隽贺月林尹红梅刘标汪彬陈薇吴迎奔王震许丽娟
Owner 湖南省微生物研究院
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