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Cellobiase genes

A technology of cellobiase and gene, applied in the field of molecular biology, can solve the problem of low cellobiase ability and achieve the effect of low solving ability

Inactive Publication Date: 2010-09-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention provides a gene encoding cellobiase, which can be highly expressed in Trichoderma reesei, and solves the problem that the ability of Trichoderma reesei to produce cellobiase is low

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0020] Cloning of embodiment 1 Aspergillus niger cellobiase gene

[0021] 1. Extraction of total RNA from Aspergillus niger

[0022] Aspergillus niger (Aspergillus niger CBS 513.88) was inoculated on PDA medium and cultured at 30°C for 7 days until the spores matured. The spore suspension was prepared and inoculated in the liquid enzyme-producing medium, cultured at 30° C. and 180 rpm for 2 days to obtain a bacterial suspension for extracting total RNA.

[0023] Liquid enzyme-producing medium: wheat bran 50g / L, heated and boiled for about 1 hour, filtered with 4-8 layers of gauze, and the filtrate was added to (NH4) 2 5O 4 10g / L, KH 2 PO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5 g / l.

[0024] The extraction steps are as follows: 1. After measuring the weight of the 1.5ml centrifuge tube, pour the bacterial suspension into centrifugation (10000rpm, 30s), discard the supernatant, and measure the weight until the bacterial weight is 100mg. 2. Use a sterilized glass rod to transfer t...

Embodiment 2

[0032] The construction of embodiment 2 Trichoderma reesei high-efficiency expression cassette

[0033] 1. Extraction of Trichoderma reesei Genomic DNA

[0034] Trichoderma reesei (ATCC 56765) was inoculated on PDA medium and cultured at 30°C for 7 days until the spores matured. The spore suspension was prepared and inoculated in the liquid seed medium, and cultured at 30°C and 180rpm for 2 days for the extraction of genomic DNA.

[0035] The above seed medium formula is: containing (g / l) glucose 20, (NH4) 2 SO 4 5. KH 2 PO 4 15. MgSO 4 0.6, CaCl 2 0.6, FeSO 4 ·7H 2 O 0.005, MnSO 4 ·H 2 O 0.0016, ZnSO 4 ·7H 2 O 0.0014, CoCl 2 0.002 (pH5.5).

[0036]In this example, the CTAB method was used to extract the genomic DNA of Trichoderma reesei. First, the cultured Trichoderma reesei cells were collected by centrifugation, and washed three times with physiological saline to remove the pigment. Transfer the mycelia into a mortar, grind with liquid nitrogen, add 400...

Embodiment 3

[0063] Embodiment 3 Construction of Hygromycin B Phosphotransferase Expression Cassette

[0064] 1. Isolation of Trichoderma reesei Ppki1 promoter sequence

[0065] According to the Ppki1 promoter sequence published on GeneBank (GI: 170552), using the extracted Trichoderma reesei genomic DNA as a template, PCR amplification was performed with upstream primer P3 and downstream primer P4 to isolate Ppki1.

[0066] The upstream primer P3, whose nucleotide sequence is shown in SEQ ID NO.11, contains EcoR I, Bgl II restriction sites;

[0067] The downstream primer P4 has a nucleotide sequence such as SEQ ID NO.12, which contains a Sac I restriction site.

[0068] The PCR reaction conditions were 94°C for 5min; 94°C for 50s, 70°C for 1min30s, 30 cycles; 72°C for 10min. The size of the product obtained by PCR amplification was 758bp. The sequencing results showed that the PCR product had a frameshift mutation of one base compared with the sequence in GeneBank, and the base sequence...

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Abstract

The invention discloses novel cellobiase genes from Aspergillus niger. A base sequence of the cellobiase genes is shown as SEQ ID NO:1. The invention also discloses an expression cassette containing the cellobiase genes, a recombinant vector and transformants. The genes can perform high-efficiency expression in Trichoderrria reesei, and the cellobiase activity of the transformants can reach 19.4 times of original strains.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a cellobiase gene. Background technique [0002] Trichoderma reesei (Trichoderma reesei) is a commonly used cellulase production strain, and the cellulase complex system it produces includes: endo-β-glucanase (endoglucanase, EG, EC3.2.1.4) , exo-β-glucanase (exoglucanase) or cellobiohydrolase (cellobiohydrolase, CBH, EC3.2.1.91), cellobiase (EC3.2.1.21, also known as β-glucosidase β -glucosidase) and other components (Nevalainen, H.and M. 1995). Cellulase must rely on the synergy between different components in the process of hydrolyzing cellulose into glucose. [0003] The cbh1 gene of Trichoderma reesei is regulated by a strong promoter, and its expression level is relatively high. Usually, the proportion of CBHI enzyme protein in the total secreted protein accounts for more than 60% (Durand, Clanet et al. 1988). This feature of the cbh1 promoter has important application ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/63C12N1/15C12R1/645C12R1/885
Inventor 王冰冰杜风光夏黎明
Owner ZHEJIANG UNIV
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