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Bacillus marinus and method for producing catalase by using same

The technology of marine bacillus and catalase is applied in the field of marine bioengineering and achieves the effects of low cost, simple preparation process, and improved expression and activity

Active Publication Date: 2013-04-24
SECOND INST OF OCEANOGRAPHY MNR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the strains authorized by bacterial fermentation to produce catalase patents are isolated from soil samples.

Method used

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  • Bacillus marinus and method for producing catalase by using same
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Isolation and screening of Iheyensis marine bacillus (O.iheyensis) CGMCC No.6042

[0025] Use NaOH or HCl to fine-tune the pH of 4.5mL natural seawater to 7.2, add 0.75% (w / v) agar and sterilize at 121°C for 20 minutes. Sterilize 4 μL of 30% hydrogen peroxide, mix well, cool and solidify to make seawater semi-solid medium. Under sterile conditions, add 4.5mL of collected seawater samples to the seawater semi-solid medium, as well as filter-sterilized carbon and nitrogen sources, and the final concentration (w / v) is peptone 0.25%, yeast extract 0.01% and casamino acids 0.25%. Shake enrichment culture at 28°C for about 4 days, dilute the enrichment solution to an appropriate multiple and spread it on a plate, pick a single clone and store it on a slant.

[0026] The bacterial strain isolated by the above method is transferred to natural seawater liquid medium for shake flask fermentation, and the catalase activity re-screening is carried out by spectrophotomet...

Embodiment 2

[0029] Example 2 Morphological identification and biological characteristics of Iheyensis marine bacillus (O.iheyensis) CGMCC No.6042

[0030] Iheya Ridge Marine Bacillus (O.iheyensis) CGMCC No.6042 was inoculated in natural seawater liquid medium, and 20g / L agar could be added to prepare solid medium. After cultivation, it was identified that the bacteria has the following characteristics: (1) Colony morphology: the colony is round, with smooth surface, flat edges, slightly raised, milky white; (2) Cell morphology: Gram-positive bacteria, under the cell microscope (Olympus, BX40) exhibits motility and is rod-shaped; (3) Physiological and biochemical characteristics: aerobic growth; oxidase positive; hydrolysis of escin, Tween 40 or Tween 60, non-hydrolysis of Tween 80, DNA or Urea; does not produce indole; can use glucose, mannose, fructose or maltose to produce acid, but cannot use arabinose or rhamnose to produce acid; to amoxicillin, ampicillin, chloramphenicol, erythromyc...

Embodiment 3

[0031] Example 3 PCR Amplification and Sequencing of the 16S rRNA Gene of O.iheyensis CGMCC No.6042

[0032] Inoculate O.iheyensis marine bacillus (O.iheyensis) CGMCC No.6042 in natural seawater solid medium, pick a ring of bacteria directly from the slope, add 200 μL of sterile water to fully suspend the bacteria, and bathe in water at 100°C for 30 minutes , centrifuged at 12000rpm for 2 minutes and immediately ice-bathed, and the supernatant was used as a template for PCR. A pair of universal primers for amplifying the 16S rRNA gene are as follows:

[0033] Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0034] Reverse primer: 5'-GGTTACCTTGTTACGACTT-3';

[0035] The above primers respectively correspond to bases 8-27 and bases 1510-1492 of the 16S rRNA gene of Escherichia coli. The PCR reaction system (50 μL) is: 5 μL of 10× buffer, 1 μL of 10 mM dNTPs, 1 μL of each 4 μM primer, 0.5 μL of Taq enzyme, 0.5 μL of template DNA, and 41 μL of sterile pure water. The PCR reaction...

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Abstract

The invention relates to an ocean source catalase high-yielding strain and a method for producing catalase by using the strain. One strain of Oceanobacillus iheyensis is obtained by directionally enriching and sieving from an ocean water body through adopting a semi-solid culture medium; and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 6042. The strain is fermented to obtain the catalase and the enzyme activity reaches 8677U / mg. A preparation process of the catalase is simple; the enzymatic activity is high and the cost is low; and the catalase is suitable for being widely applied to the fields of foods, spinning, papermaking, environmental friendliness and the like. Therefore, an enzyme producing strain and a fermentation method disclosed by the invention have wide industrial application values and obvious economic benefit prospects.

Description

technical field [0001] The invention belongs to the technical field of marine bioengineering, and in particular relates to a catalase high-yielding bacterium derived from marine sources and a method for producing catalase by using the bacterium. Background technique [0002] Catalase is a terminal oxidase that widely exists in animals, plants and microorganisms. It converts hydrogen peroxide (H 2 o 2 ) as the substrate, which is decomposed into water and oxygen. Catalase can quickly remove oxygen free radicals produced in the oxidation-reduction reaction of biological metabolism and prevent cells from being damaged by strong oxidants. It is an important antioxidant protection system in organisms. In recent years, with the H 2 o 2 Widely used in industries such as textiles, papermaking, food, medicine and environmental protection, the market demand for catalase is also extensive and growing. [0003] In paper and textile industry, catalase can be used for H 2 o 2 Deoxy...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/08C12R1/01
Inventor 霍颖异许学伟王春生吴敏张心齐潘杰
Owner SECOND INST OF OCEANOGRAPHY MNR
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