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Novel heat-resisting halophilic lipase and extreme halophile for generating same
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A technology of halophilic bacteria and lipase, applied in the field of new halophilic and heat-resistant lipase
Inactive Publication Date: 2010-06-02
JIANGNAN UNIV
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However, there is no domestic literature report on the research on halophilic and heat-resistant lipase
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Embodiment 1
[0050] Embodiment 1: Screening of producing halophilic lipase strain
[0051] Transplant and activate the strains, culture at 37°C, pick a single colony and plant it on an olive oil-rhodamine B plate for 7-14 days at 37°C, select the colony with a fluorescent circle and separate it by streaking to obtain a single colony, inoculate Stored in slant medium, fermented in shake flasks and re-screened. see results Image 6 :
Embodiment 2
[0052] Embodiment 2: the mensuration of lipase activity
[0053] The determination of lipase enzyme activity adopts colorimetric method, the principle is: p-NPB is decomposed under the catalysis of lipase to generate a molecule of p-nitrophenol, p-nitrophenol has the maximum absorption peak at 410nm, by measuring the reaction solution at The absorbance value at 410nm can be used to quantitatively measure the oxidation amount of p-NPB, so as to calculate the enzymatic activity unit of lipase.
[0054] The determination method is carried out with reference to the literature, as follows:
[0055] Solution A: Dissolve 176 μL p-NPB in 10 mL isopropanol, store at 4°C
[0056] Solution B: pH 8.0, 50mmol / L Tris-HCl buffer
[0057] When in use, mix solution A and solution B at a volume ratio of 1:9 to prepare 3.6 mL of substrate reaction solution, add 400 μL of crude enzyme solution, react at 37°C for 10 min, and measure the absorbance at 410 nm.
[0058] Definition of 1 enzyme acti...
Embodiment 3
[0060] Example 3: Identification of 16s rDNA strains
[0061] Take the fresh bacterial solution in the exponential growth phase, collect the bacterial cells by centrifugation, and extract the genomic DNA according to the genome extraction kit. Amplification primers are general primers for archaea,
[0072] PCR amplification conditions: denaturation at 95°C for 1min, annealing at 56°C for 45s, extension at 72°C for 90s, 50μL system, 35 cycles. Purification of PCR amplification products was carried out according to the instruction of Shanghai Sangon Biotechnology Co., Ltd. Small Volume Gel Recovery PCR Product Pu...
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Abstract
The invention belongs to the field of biotechnology, which particularly relates to a novel heat-resisting halophilic lipase and an extreme halophile JNPH-3 for generating the same, wherein the extreme halophile JNPH-3 is obtained by screening from a salt lake in Xilinhot city, Inner Mongolia. The strain of the extreme halophile is preserved in China General Microbiological Culture Collection Center, and the preservation Number is CGMCC. No.2462. The most suitable pH value for the growth of the strain is 7.0, and the most suitable temperature for the growth of the strain is 45 DEG C. The novel heat-resisting halophilic lipase generated by the strain belongs to an important industrial enzyme for commercial use, and can be widely used in the fields of food, chemicals, medicines, and the like.
Description
technical field [0001] The invention relates to an extreme halophilic bacterium, in particular to an extreme halophilic bacterium producing a new type of halophilic and heat-resistant lipase screened from a salt lake in Xilinhot, Inner Mongolia, and a new type of halophilic and heat-resistant fat produced by the strain An enzyme belongs to the field of biotechnology. Background technique [0002] Extremophiles have unique gene types, special physiological mechanisms and special metabolites. As marginal life phenomena on the earth, extremophiles are quite intriguing. They will give people many important enlightenments in terms of the origin of life and system evolution. The principle of life behavior will also expand people's concept, and the principle of extremophile existence has great application value. The special mechanism and special products of extremophiles will make some new biotechnology methods possible and lay the foundation for high-efficiency, low-cost new biot...
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