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Aroma-producing pichia guilliermondii for producing beta-glucosidase and application

A technology of glucosidase and fragrant season, which is applied in the direction of glycosylase, enzyme, enzyme, etc., can solve the problem of no reported production of β-glucosidase, etc., and achieve simple operation, fast growth and simple culture conditions Effect

Active Publication Date: 2020-07-31
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No β-glucosidase-producing strains of Pichia mongolica have been reported

Method used

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  • Aroma-producing pichia guilliermondii for producing beta-glucosidase and application
  • Aroma-producing pichia guilliermondii for producing beta-glucosidase and application
  • Aroma-producing pichia guilliermondii for producing beta-glucosidase and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Pichia mongoliana GXDK6 isolation and identification

[0052] (1) Sampling was carried out from mangrove soil sediments in Beihai City, Guangxi Province. After the samples were dissolved in sterile water, they were shaken and cultured on a shaker at 37°C for one hour, and then diluted to 10% by gradient with sterile distilled water. -1 、10 -2 、10 -3 、10 -4 、10 -5 , take 10 respectively -2 ~10 -5 100 μL of the diluted bacterial solution was spread on the YPD solid medium, cultured at 37°C for 2 to 3 days, and single colonies with milky white, slightly convex, moist, neat edges, regular round shape and smooth surface were picked and streaked on the plate 2 ~3 times a single colony was obtained.

[0053] (2) Activation of Pichia mongolianus GXDK6

[0054] The strains taken out from -80°C were inoculated and streaked in GBM solid medium, and cultured at a constant temperature of 30°C for 48h.

[0055] (3) Whole-genome sequencing of Pichia mongoliana GXDK6 ...

Embodiment 2

[0085] Example 2 Gas Chromatography-Mass Spectrometry Detection of Volatile Fragrance Substances

[0086] (1) Determination of fermentation flavor

[0087] Use GBM medium and 2% of the mass volume percentage of the aroma-producing fermentation medium as the inoculum amount, culture at 30°C and 200rpm for 1-7 days, and smell the flavor of the liquid fermentation liquid. The liquid fermentation liquid has sweet and fruity aromas and other special fragrance.

[0088] (1) Detection of special aroma metabolites

[0089] The samples of GBM medium 11, 24h and aroma production medium 24h, 72h were treated and analyzed by gas chromatography-mass spectrometer. GC-MS analysis conditions: GC conditions: inlet temperature 250°C, carrier gas helium (He), flow rate 2m L / min. Injection volume 1 μL, splitless injection, chromatographic column (30m×250μm×0.25μm), heating program: 40°C constant temperature for 4min, temperature rising to 210°C at a speed of 10°C / min, and keeping for 6min. MS...

Embodiment 3

[0101] Embodiment 3 pNP colorimetric assay β-glucosidase enzymatic properties

[0102] (1) Drawing of pNP standard curve

[0103] Prepare pNP standard solutions of different concentrations according to the following table, each concentration is 3 in parallel, mix well, draw 200 μL from the pNP standard solutions of each concentration into EP tubes, add 50 μL 2mol / LNa respectively 2 CO 3 Mix the solution evenly, put 200 μL of the above solution in the enzyme label plate, and read the OD of each concentration 410 Absorbance value, then the pNP concentration as the abscissa, the OD 410 The value is used as the ordinate to draw the standard curve of pNP.

[0104] Numbering 0 1 2 3 4 5 6 7 8 Distilled water (μL) 1000 995 990 985 980 975 970 965 960 10μmol / mLpNP(μL) 0 5 10 15 20 25 30 35 40 pNP concentration (μmol / mL) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

[0105] (2) Enzyme activity assay

[0106] Take 1ml of fer...

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Abstract

The invention belongs to the technical field of industrial microorganisms, and relates to aroma-producing pichia guilliermondii for producing beta-glucosidase and an application. The aroma-producing pichia guilliermondi GXDK6 for producing beta-glucosidase is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 25, 2018, wherein the preservation number of the strain is CGMCC No. 16007. The strain provided by the invention is the aroma-producing pichia guilliermondii strain which is reported for the first time and is used for producing beta-glucosidase. The pichia guilliermondii provided by the invention can utilize pentose and hexose as unique carbon sources to produce beta-glucosidase. The pichia guilliermondii provided by the invention can be fermentedfor a long time to produce fragrance. The pichia guilliermondii provided by the invention can utilize organic matters, is salt-resistant, can tolerate high-concentration heavy metals and effectivelyadsorb the heavy metals, can adapt to a wide pH range, and expands the environmental conditions for producing beta-glucosidase.

Description

technical field [0001] The invention belongs to the technical field of industrial microbes, and relates to a Pichia mongolica producing beta-glucosidase and its application. Background technique [0002] In 1837, Liebig and Wohler first discovered a cellulase in bitter almond juice that can hydrolyze the non-reducing β-D-glucose bond bound to the terminal and release β-D-glucose and the corresponding ligand. This enzyme is named β-glucosidase (EC 3.2.1.21), also known as β-D-glucoside glucohydrolase, amygdalinase, gentiobiase, cellobiase (cellobias, CB or β- G). β-glucosidase has great application value in industrial production. First of all, β-glucosidase is an important component in the cellulolytic enzyme system, and it is also a key enzyme in the last step in the process of hydrolyzing cellulose. Secondly, β-glucosidase can be used as a key enzyme for aroma improvement or enhancement, which can hydrolyze flavor precursors more gently and quickly, release bound volatil...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N9/42B01J20/22C02F3/34C02F101/20C02F101/22C12R1/84
CPCB01J20/22C02F3/34C02F2101/20C02F2101/206C02F2101/22C12N9/2445C12Y302/01021C12N1/165C12R2001/84
Inventor 蒋承建莫雪艳阎冰何升惠秦妍李全文蔡杏华于苒季凯欧倩
Owner GUANGXI UNIV
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