Achromobacter xylosoxidans strain for producing carboxymethyl cellulase

A technology for carboxymethyl cellulose and cellulose degradation, which is applied in the field of bioengineering and can solve problems such as lack of alkaline cellulase production strains

Active Publication Date: 2014-12-24
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] More importantly, there is a lack of alkaline cellulase production strains that have both production benefits and environmental protection benefits, which can degrade cellulose and degrade environmental pollutants at the same time.

Method used

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  • Achromobacter xylosoxidans strain for producing carboxymethyl cellulase
  • Achromobacter xylosoxidans strain for producing carboxymethyl cellulase
  • Achromobacter xylosoxidans strain for producing carboxymethyl cellulase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Screening, isolation and identification of strains

[0026] The first step: sampling

[0027] Compost samples in the suburbs of Wuming County, Nanning City, Guangxi Zhuang Autonomous Region.

[0028] Step 2: Primary screening, secondary screening and purification

[0029] Take 1g sample, add 10ml sterile distilled water, shake well, stand for 30 minutes, take the supernatant and dilute 10 5 CMC-Congo red plate screening medium was coated and inoculated, cultured at 30°C for 72 hours until colonies grew, picked the colonies with larger transparent hydrolysis circles, inoculated with CMC liquid fermentation medium, cultured in a shake flask at 37°C, 200rpm for 24 hours, and measured The alkaline CMC enzyme activity of the culture medium. Select the most vigorous culture flask, inoculate the CMC-Congo red plate screening medium with the bacterial solution streaked, and purify the strain 3 times, and finally obtain a high-yielding strain of alkaline cellulase, named 7B, ...

Embodiment 2

[0035] Example 2 Detection of cellulase production by strains

[0036] according to figure 1 with figure 2 As shown in the optimum temperature and pH for growth, strain 7B was inoculated with CMC fermentation medium and cultivated, and the enzyme production curve was drawn. The results are as follows Figure 4 Shown. The preparation method of the crude enzyme solution is as follows: the activated strain is inoculated with CMC fermentation medium, cultivated at 30°C, 200rpm for 48h, the culture solution is taken, centrifuged at 4000rpm at 4°C for 10min, the supernatant is taken, and 55% (NH 4 ) 2 SO 4 Precipitate, centrifuge at 10,000 rpm for 10 min at 4°C, collect the precipitate, add the same volume of the culture solution to the pH 8.0, 0.1 mol / L Tris-HCl buffer to dissolve. Using CMC sodium salt as a substrate, the activity of endocellulose, that is, CMC enzyme activity, was determined. The enzyme reaction system contains 0.5 ml of crude enzyme solution, 1% CMC sodium salt, a...

Embodiment 3

[0040] Example 3 Treatment of BTEX and refractory chelating agent

[0041] This example was performed at the laboratory level. A 10-fold diluted LB medium with 2% CMC cellulose was used to simulate a sewage water body rich in organic matter. Respectively: adding 0.02% ethylbenzene to simulate BTEX polluted industrial wastewater; adding 0.1% IDS (imino disuccinic acid) to simulate refractory chelating agent polluting water; adding 0.1% endosulfan to simulate refractory pesticide polluting water. The experiment used 100mL culture solution, inoculation amount 1%, 30℃, rotating speed 180rpm shake flask culture for 48 hours. Sampling every 4 hours to detect residual COD and pollutants. Among them, the determination of ethylbenzene residues adopts n-hexane extraction + gas chromatography, wherein the chromatographic column is HP-Innoax capillary column, column temperature is 90℃, column flow rate is 1mL / min, and carrier gas is N 2 ; COD determination uses potassium dichromate oxidati...

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Abstract

The invention discloses a bacterium 7B capable of producing cellulase separated and screened from stockpile manure. The bacterium is collected by China Center for Type Culture Collection, and the collection number is CCTCC NO:M2013365. The strain 7B is Gram-negative bacilli, and forms a smooth, wet, lustrous, offwhite bacterial colony with regular edge, of which the diameter is 0.5-1mm, on the LB (Langmuir-Blodgett) culture medium. A BIOLOG microorganism identification system and 16s rDNA sequence analysis are utilized to identify that the strain is Achromobacter xylosoxidans. A CMC (carboxymethyl cellulose) fermentation culture medium is used for culturing to obtain the 0.183U / mL carboxymethyl cellulase activity, and the Achromobacter xylosoxidans strain is hopeful to be used for cellulase production.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and particularly relates to a cellulase-producing Achromobacter xylosoxidans 7B obtained by screening, separating, purifying, and obtaining cellulase from cattle manure compost samples. Background technique [0002] After entering the 21st century, as the prospect of fossil fuels tends to be exhausted, excessive carbon emissions, environmental pollution and ecological imbalances have become prominent, people are increasingly turning their attention to renewable clean new energy. Lignocellulose is one of the excellent resources. The use of lignocellulose to produce fuel ethanol has become a promising clean energy technology. Lignocellulose is abundant in agricultural production, but its value has not been deeply explored. It is estimated that the global cellulose production is 660-8800 billion tons per year. As a large agricultural country, my country also produces a large amount of lignocell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12R1/025
Inventor 芦志龙陈东张穗生吴仁智陆琦黄日波
Owner GUANGXI ACAD OF SCI
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