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Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation

A cellulase and Penicillium oxalate technology, applied in the biological field, can solve the problems of high production cost, low cellulase activity and the like

Active Publication Date: 2013-01-16
广西鼎乐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still problems of low cellulase activity and high production costs

Method used

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  • Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation
  • Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation
  • Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] In Example 1, the Congo red-Avicel solid medium used in the isolation and screening of mutant strains was (g / L): KH 2 PO 4 2g, (NH 4 ) 2 SO 4 1.4g, CaCl 2 0.3g, MgSO 4 .7H 2 O 0.5g, urea 0.3g, Yeast extract 0.8g, Peptone 2g, glucose 2g, Avicel PH-1013g, gelatin 2g, agar 20g, Congo red 0.2g, trace elements 0.05mL, pH 5.0, sterilized at 121°C for 30min.

[0049] When rescreening mutant strains, use liquid screening medium (g / L): KH 2 PO 4 4g, (NH 4 )2 SO 4 2.8g, CaCl 2 0.6g, urea 0.6g, MgSO 4 .7H 2 O 0.6g, wheat bran 20g, Avicel 30g, Tween-802mL, trace elements 0.1mL (the components of trace elements and the final concentration in the culture medium are respectively 5.0mg / LFeSO 4 ·7H 2 O,1.6mg / L MnSO 4 ·H 2 O,1.4mg / L ZnSO 4 ·7H 2 O, 2.0mg / L CoCl 2 ), pH 5.0, sterilized at 121°C for 30min.

[0050] When performing liquid shake flask re-screening on mutant strains, use the method of plate cutting, inoculate mycelium and spores (mycelia and spores with a ...

Embodiment 2

[0052] In Example 2, the enzymatic activities of various cellulases were measured by the following method.

[0053] 1) Glucose concentration standard curve

[0054] Prepare 1mg / mL glucose standard solution with deionized water. Add the solution into the test tube according to the formula in Table 1, mix well, develop color in boiling water for 5 minutes, and cool in cold water. Take 200 μL of the solution, add it to a 96-well microtiter plate, measure the absorbance at 540 nm, and draw a glucose standard curve. The regression equation is Y=3.1803X—0.0417, and the variance R 2 is 0.9992.

[0055] Table 1 Preparation of glucose concentration standard samples

[0056]

[0057] 2) Standard curve of p-nitrophenol (p-NP) concentration

[0058] Prepare 1 mg / mL p-NP standard solution with deionized water. Add the solution to the test tube according to the formula in Table 2. After mixing, draw 200 μL of the solution, put it in a 96-well microtiter plate, measure its absorbanc...

Embodiment 3

[0183] Example 3. Preparation of cellulase preparation (cellulase) using Penicillium sp. mutant strain EU2106

[0184] 1. Preparation of fermentation medium

[0185] Prepare an optimized medium for pH 5.5: KH 2 PO 4 4g, (NH 4 ) 2 SO 4 4g, CaCl 2 0.6g, MgSO 4 .7H 2 O0.6g, Tween-802mL, trace elements 0.1mL (the components of trace elements and the final concentration in the culture medium are respectively 5.0mg / L FeSO 4 ·7H 2 O,1.6mg / L MnSO 4 ·H 2 O,1.4mg / L ZnSO 4 ·7H 2 O,2.0mg / LCoCl 2 ), wheat bran 40g, Avicel 10g, pH adjusted with 2M HCl aqueous solution or 2M NaOH aqueous solution; distilled water to 1L; sterilized at 121°C for 30min.

[0186] 2. Preparation of spore solution

[0187] (1) Prepare PDA solid medium and sterilize it at 112°C for 20 minutes.

[0188] (2) Inoculate the Penicillium sp. mutant strain EU2106 on a PDA solid medium plate and culture it in a constant temperature incubator at 28°C for 5 days. Wash the spores with a certain volume of 0.9%...

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Abstract

The invention discloses a penicillium sp. mutant strain and application of the penicillium sp. mutant strain to cellulase preparation. The invention provides a strain of penicillium oxalicum EU2106 with the preservation number being CGMCC (China General Microbiological Center Culture Collection Center) No.6471. The invention also provides a method for producing cellulase or a cellulase preparation. The method comprises the following steps that the penicillium sp. mutant strain EU2106 is cultured for a certain time on a basic or optimized liquid culture medium, culture substances are collected, thalli are removed through centrifugation, and then, obtained supernate can be used as the cellulase preparation. Experiments prove that the penicillium oxalicum EU2106 obtained through the mutation can be used for producing the cellulase preparation, the preparation can be used for effectively hydrolyzing sugarcane slag paper pulp, and in addition, glucose is major ingredients in the final hydrolysis products. The cellulase preparation can be used for simultaneous saccharification and fermentation of sugarcane slag paper pulp for alcohol production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant strain of Penicillium and its application in the preparation of cellulase. Background technique [0002] Lignocellulose is the most widely distributed and abundant renewable organic resource on the earth. At present, lignocellulose resources have not been effectively utilized. Fuel ethanol refers to a new type of fuel used in engines after absolute ethanol has been treated with a denaturant. The common raw materials for its production are corn starch, tapioca starch, and sugarcane juice. These raw materials are converted into ethanol under the action of enzymes or microorganisms. The use of microorganisms to convert cellulose into glucose and then ferment to produce fuel ethanol is of great significance for solving the problems of competition with people and land caused by the production of fuel ethanol with grain or carbohydrates as raw materials. [0003] Since my countr...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12P19/14C12P19/02C12P19/12C12P7/06C12R1/80
CPCY02E50/17Y02E50/10
Inventor 冯家勋农清栋刘君梁秦秀林段承杰张政黄妹平蓝健益卢业飞吕芳贤张颖黄叶萍
Owner 广西鼎乐生物科技有限公司
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