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A Cellulase Gene and Its Application from Sclerotinia hypertrophicus Mulberry

A technology of cellulase and sclerotinia, which is applied in the field of bioengineering, can solve problems such as poor thermal stability, difficulty in strain selection, and low enzyme production

Inactive Publication Date: 2016-05-11
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main factors restricting the large-scale application of cellulase are: (1) difficulty in strain selection and low enzyme production; (2) poor thermal stability

Method used

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  • A Cellulase Gene and Its Application from Sclerotinia hypertrophicus Mulberry
  • A Cellulase Gene and Its Application from Sclerotinia hypertrophicus Mulberry
  • A Cellulase Gene and Its Application from Sclerotinia hypertrophicus Mulberry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Gene full-length cloning

[0024] (1) First, design primers for amplification based on the S. sclerotiorum cellulase gene reported on the S. sclerotiorum datebase (http: / / www.broadinstitute.org / annotation / genome / sclerotinia_sclerotiorum / MultiHome.html). The upstream primer is: 5'-ATGCGTTTCGCAAACTTCAT-3', the downstream primer is: 5'-TTATGCTACATAGCCGACAA-3', and the primers were synthesized in Nanjing GenScript Biotechnology Co., Ltd. The hyphae of [Ciboriashiraiana (P.Hennings) Whetzel] were cultured in YPD liquid medium for 5 days, the culture conditions were 25° C., 180 rpm. The mycelium was filtered by vacuum filtration, rinsed twice with PBS, RNA was extracted according to the instructions of RNAPlus from Takara Company, and reverse-transcribed into cDNA, which was used as a PCR template to amplify the full length of the sscellulase gene, and the recovered product was transformed into Escherichia coli DH5ɑ competent Cells, the obtained positive clones we...

Embodiment 2

[0027] Example 2: Transformation of P.pastorisGS115 strain and positive selection

[0028] The pPIC9K-sscellulase plasmid was extracted, and linearized by single digestion with SacI, and the pPIC9K empty plasmid also digested with SacI was used as a control. Then pPIC9K-sscellulase and pPIC9K dephosphorylated with dephosphorylase were transformed into P. pastorisGS115 strain by electric shock transformation method.

[0029] The specific operation steps for transforming P.pastorisGS115 are as follows: (1) Preparation of P.pastorisGS115 competent cells:

[0030] Inoculate P.pastorisGS115 in 100mL LYPD medium, culture at 30°C, 240rpm to OD600=1.2-1.5, 6000rpm, 5min, collect the bacteria by centrifugation, wash the bacteria with 30mL, 10mL pre-cooled sterile water successively, 6000rpm / Centrifuge for 1 min to remove the supernatant, wash the cells with 2.5mL of 1mol / L sorbitol solution, and finally suspend the obtained cells in 0.5mL of pre-cooled 1mol / l sorbitol, and dispense 8...

Embodiment 3

[0036] Example 3: Induced expression of target gene

[0037] 100 mL of GS115 / pPIC9K-sscellulase4 strain was induced for 144 hours, and the supernatant was dried by a freeze dryer for 48 hours. Determination method The DNS (3,5-dinitrosalicylic acid) method is used to measure the filter paper enzyme activity of the enzyme, and the enzyme activity is defined as: 1 mg of glucose produced per milliliter of enzyme solution for 1 min is a unit. The specific method is as follows: draw 1% glucose 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.4mL in test tubes 1-8, and then use the buffer solution as lemon juice with pH=5.0 Acid-sodium citrate buffer was made up to 2 mL in each tube. Add 6 pieces of 1*1cm 2 (Xinxing, Hangzhou), react at 45°C for 30min, quickly add 3mL DNS solution (3,5-dinitrosalicylic acid 10g, NaOH 2g, potassium sodium tartrate 20g, phenol 2g, sodium sulfite 5g, 1000mL water, dark Store in place for 7 days.) in each test tube No. 1-8. Place the test tube in bo...

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Abstract

The invention clones an acid-producing cellulase gene (sscellulase) sequence carried by the Ciboriashiraiana (P.Hennings) whetzel, and discloses a cloning method and application value. The full-length gene is 1170 bp, and 390 amino acids are coded, pI is equal to 4.25, and the optimum pH is 5.0, the optimum temperature is 45 DEG.C. The gene has high eukaryotic expression cellulase activity and effects of decomposing cellulose as monosaccharide such as glucose, and can be applied to the gene engineering of the cellulase.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an acid-producing cellulase gene (sscellulase) carried by Sclerotinia mulberry hypertrophic sclerotinia [Ciboriashiraiana (P.Hennings) Whetzel] and its application. Background technique [0002] Cellulose is the most abundant natural organic matter in the world. It is a renewable resource with the advantages of wide sources, many varieties, and renewable utilization. It accounts for more than 50% of the carbon content in the plant kingdom. my country is very rich in cellulose raw materials, and the annual output of crops and hulls alone reaches more than 700 million tons. Cellulase is a general term for a class of multi-enzyme systems that can degrade cellulose into glucose. They act synergistically to decompose cellulose to produce oligosaccharides and cellobiose, which are finally hydrolyzed into glucose. In addition to being used as a carbon source for the production of alcohol,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N15/70C12N9/42
Inventor 余茂德吕蕊花赵爱春李军刘长英鲁成王茜龄吴存容裴汭超
Owner SOUTHWEST UNIV
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