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A kind of gladiolus Burkholder strain and its fermentative method for producing alkaline lipase

A technology of Holder's bacteria and lipase, which is applied in the field of Burkholder's strain and its fermentation to produce alkaline lipase, can solve the problems of limited lipase activity and stability, achieve low production cost, increase yield, The effect of stabilizing enzyme production

Active Publication Date: 2018-03-20
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the non-aqueous enzyme-catalyzed reaction has the above advantages and the advantages of convenient post-catalysis treatment, the activity and stability of lipase are limited by traditional organic solvents.

Method used

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  • A kind of gladiolus Burkholder strain and its fermentative method for producing alkaline lipase
  • A kind of gladiolus Burkholder strain and its fermentative method for producing alkaline lipase
  • A kind of gladiolus Burkholder strain and its fermentative method for producing alkaline lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening and identification of alkaline lipase-producing strains

[0033] 1. Preparation of Oriented Screening Plates

[0034] According to the characteristics that Burkholderia can use special metabolites and resistance to antibiotics, an improved TB-T plate is designed for directional screening, and the positive rate of screening Burkholderia can be as high as 100%. The detailed formula is as follows: 1L modified TB-T directional screening plate contains 2g of glucose, 0.5g of L-asparagine, NaHCO 3 0.5g, KH 2 PO 4 0.25g, MgSO 4 ·7H 2 O 0.05g, trypan blue 0.05g, tetracycline 0.01g, ampicillin 0.15g, agar powder 15g.

[0035] 2. Preparation of Identification Plates

[0036] A 1 L rhodamine B identification plate contains 5 g of peptone, 3 g of yeast extract, 8 g of NaCl, 4 mL of rhodamine B solution (0.1%, W / V), and 10 mL of soybean oil emulsion.

[0037] The preparation method of the soybean oil emulsion is as follows: 3mL soybean oil and 9mL polyvin...

Embodiment 2

[0049] Embodiment 2: Utilize BPS-1 bacterial strain and optimize medium fermentation to produce lipase

[0050] (1) Seed culture: After the BPS-1 strain is activated, it is inoculated in liquid LB medium, and cultivated at 30-35°C for 12-18 hours to obtain a seed culture solution;

[0051] (2) Fermentation culture: inoculate the seed liquid into the initial fermentation medium (as described in Example 1) by the amount of 1-1.5% (V / V), after continuous cultivation for 72 hours, 8000r / mim, centrifugal 10min, measure lipase activity.

[0052] The carbon source (glucose, sucrose, molasses, dextrin, maltose, malt extract, starch) and nitrogen source (peptone, yeast extract, corn steep liquor, urea, NaNO) of the fermentation medium were studied by single factor replacement method. 3 , NH 4 SO 4 ) and inducers (olive oil, palm oil, corn oil, soybean oil, rice bran oil, castor oil, rapeseed oil) on the production of enzymes, the optimized components were molasses 1.0% (v / v), Palm ...

Embodiment 3

[0053] Embodiment 3: the purification of alkaline lipase

[0054] 1. The optimized fermentation broth described in Example 2 is precipitated with ethanol with a final concentration of 60% (v / v), centrifuged at 8000r / mim for 10min to remove foreign proteins, and then the final concentration is 95% (v / v) The target protein was precipitated with ethanol, and the obtained precipitate was collected after centrifugation at 8000 r / mim for 10 min, and dissolved in 0.05 mol / L piperazine buffer solution with a pH value of 9.7 to obtain a preliminary purified enzyme solution.

[0055] 2. The initially purified enzyme solution was subjected to HiTrap Q ion exchange chromatography and superdex 75 gel filtration chromatography, and further purified to obtain electrophoretic pure lipase protein (such as figure 1 Shown), the molecular weight of this lipase is about 35kDa, and the lipase specific activity after purification is 443904.8U / mg.

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Abstract

The invention relates to a high-yield alkaline lipase-producing Burkholderia strain (Burkholderia gladioli BPS‑1), which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC No.10533. The bacterial strain is derived from rotting onions, has stable enzyme production, wide sources of raw materials, and low production cost. The invention also provides a method for preparing alkaline lipase by the bacterial strain, which comprises the steps of seed cultivation, fermentation cultivation and separation and purification. The method optimizes the fermentation medium, and the prepared lipase has strong activity, up to 145.67U / mL, excellent stability in an alkaline environment, and good tolerance to short-chain alcohols. It is used in biomass energy, organic It has broad application prospects in the fields of synthesis, chiral resolution of drugs and tool enzymes.

Description

technical field [0001] The invention relates to microorganisms and enzymes, in particular to a Burkholder strain and a method for fermenting and producing alkaline lipase. Background technique [0002] Lipase (EC 3.1.1.3) is a special class of ester bond hydrolase, a biocatalyst with multiple catalytic functions, which can catalyze the synthesis reactions of substrate hydrolysis, acidolysis, alcoholysis, ammonolysis, transesterification and esterification , are widely used in food industry, paper industry, washing industry, pharmaceutical field and related industries and fields. [0003] The mechanism of the lipase-catalyzed reaction is similar to that of serine protease. The hydroxyl group on the serine residue in the active center initiates a nucleophilic attack on the carbonyl C atom of the substrate to form a tetrahedral intermediate complex, and the alcohol group falls off from the complex to form an acyl-enzyme Complex, which is a key intermediate in different catalyt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/20C12R1/01
Inventor 朱婧王青艳申乃坤朱绮霞廖思明刘海余
Owner GUANGXI ACAD OF SCI
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