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Vibrio harveyi variant capable of producing lactase and application thereof

A technology of Vibrio harveyi and lactase, applied in the field of microorganisms, to achieve a wide range of adaptation effects

Pending Publication Date: 2022-04-12
NINGBO XINUOYA MARINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is disclosed in the prior art that Vibrio harveyi can express and secrete metalloproteases, chitinases and phospholipases, etc., and there are few reports on the research on the ability of Vibrio harveyi to express lactase

Method used

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  • Vibrio harveyi variant capable of producing lactase and application thereof
  • Vibrio harveyi variant capable of producing lactase and application thereof
  • Vibrio harveyi variant capable of producing lactase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The acquisition of embodiment 1 Vibrio harveyi variant (Vibrio harveyi) bacterial strain

[0024] 1. Strain screening

[0025] Take the seawater sample stock solution brought back from Boao Port, Hainan (E110.60848, N19.186684) and coat it with LB solid medium, add 2‰ of X-Gal to the medium, and place the LB plate in a 30°C incubator Cultivate upside down for 2-4 days, then select a blue single colony from the plate and transfer it to a new LB solid medium to obtain purified strains such as figure 2 shown. Inoculate the purified strain into LB liquid medium, culture on a shaking table (30°C, 245rpm), and after 12 hours, take the bacterial liquid and add glycerol and store it in a -80°C refrigerator, and take samples at 48h and 96h to measure the activity of lactase .

[0026] The vibrio harveyi bacterial strain obtained by screening, because it produced the most obvious blue spots on the LB plate medium (containing 2‰ of X-Gal), so the bacterial strain is a lactase-...

Embodiment 2

[0027] Gene sequence analysis of embodiment 2 Vibrio harveyi variant (Vibrio harveyi) bacterial strain

[0028] 1. Identification of bacterial strains: First, the 16S rDNA of the bacterial strain was extracted by kit extraction method, amplified by PCR, general primers 806R and 16S-515F were used for PCR amplification, and the PCR reaction system was: Mix (dNTP, enzyme, Mg 2+ , 10×buffer) 25 μL, 806R primer 2 μL, 16S-515F primer 2 μL, bacterial liquid template 2 μL, sterile water 19 μL. The PCR amplification program was: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 1 minute, and 32 cycles; extension at 72°C for 10 minutes, and termination at 4°C. PCR products were identified by 1% agarose gel electrophoresis. The target PCR product was recovered and purified using the EasyPure PCR Purification Kit (Beijing Quanshijin Biotechnology Co., Ltd.). Then perform full-length sequencing, and the measu...

Embodiment 3

[0030] The optimal pH of the enzyme activity of the lactase expressed by the Vibrio harveyi strain of embodiment 3

[0031] Definition of enzyme activity unit: One enzyme activity unit (NLU) is defined as the amount of enzyme required to release 1.30 μmol / min of o-nitrophenol under the reaction conditions.

[0032] Enzyme activity assay method: o-nitrophenol β-D galactoside (ONPG) was dissolved in P-E-M buffer solution (pH value 6.5) to prepare a substrate solution with a mass fraction of 0.25%. Take 5ml of the substrate solution and add it to 1mlL of the enzyme solution, and incubate at 30°C for 10min. Add 2mL of sodium carbonate solution to develop color, and measure the light absorption value at 420nm. The content and enzyme activity of the hydrolyzed product o-nitrophenol (ONP) were calculated.

[0033] Take the enzyme solution and dilute it with buffer solution with pH of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.7 and 8.0 respectively, and measure the en...

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Abstract

The invention provides a vibrio harveyi variant capable of producing lactase, the strain number is BHNC004002, the strain is preserved in the China Center for Type Culture Collection (CCTCC) on October 11, 2021, and the preservation number is CCTCC No: M20211247. The invention also provides an application of the vibrio harveyi variant in expression of lactase. The lactase expressed by the vibrio harveyi variant has wide application ranges of enzyme activity temperature and enzyme activity PH value.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a lactase-producing Vibrio harveyi variant and its application. Background technique [0002] Lactase, scientific name β-galactoside galactohydrolase, also known as β-galactosidase, is a white powder, tasteless and odorless. Lactase catalyzes the hydrolysis of the β-galactoside bond in the β-galactoside compound under specific conditions, and hydrolyzes excess lactose in the human body into glucose and galactose, which are easily absorbed by the intestine. At present, lactase can be divided into intracellular enzymes or extracellular enzymes according to different sources. For example, lactase produced by lactic acid yeast, Aspergillus niger, Aspergillus oryzae, and rice root enzymes are all extracellular enzymes, Kluyveromyces fragilis and most bacteria produce intracellular enzymes. The optimal pH of lactase from Clavieromyces lactis is 6.0-7.0; the optimal temperature is 40...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/38C12R1/63
Inventor 曹阳丁慧田健储辉
Owner NINGBO XINUOYA MARINE BIOTECH CO LTD
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