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Penicillium decumbens engineered strain containing over-expressed beta-glucosidase and application thereof

A technology of glucosidase and Penicillium recumbentum, which is applied in the field of microbial engineering and can solve the problem that the output of β-glucosidase cannot meet the demand and the like

Inactive Publication Date: 2010-09-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Penicillium decumbens (Penicillium decumbens) has strong cellulase production capacity, but its β-glucosidase production can not meet the demand in the existing production method, and the lower β-glucosidase in its cellulase system Glucosidase content remains the key factor limiting cellulose degradation by Penicillium decumbens
The purposeful construction of engineered bacteria by overexpressing β-glucosidase is expected to overcome the above-mentioned defects. However, there are no reports of related work in the search results.

Method used

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  • Penicillium decumbens engineered strain containing over-expressed beta-glucosidase and application thereof
  • Penicillium decumbens engineered strain containing over-expressed beta-glucosidase and application thereof
  • Penicillium decumbens engineered strain containing over-expressed beta-glucosidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. β-glucosidase overexpression vector pTBG-1 transforms Penicillium decumbens JU-A10

[0030] Firstly, Double-joint PCR (DJ-PCR) technology (Jae-Hyuk Yu et al. (2004). Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genetics and Biology. 41: 973- 981) Cloning of the bgl1 cDNA gene of Trichoderma reesei (GenBank sequence U09580.1). Using B1S / B1A, B2S / B2A, and B3S / B3A as primer pairs and extracted Trichoderma reesei QM9414 (ATCC 26921) chromosomal DNA as a template, amplify the 3 exon fragments of the bgl1 gene, and recover 89bp from the gel , 1760bp and 442bp amplified fragments, and then use chimeric primers to assemble the three recovered fragments. Finally, B1S and B3A were used as primers, and the self-assembled PCR product was used as a template to amplify bgl1 cDNA. The PCR product of about 2.3kb was recovered from the gel, ligated into pMD18-T, and sent to Shanghai Biological Engineering Co., Ltd. for seq...

Embodiment 2

[0058] Example 2. PCR verification of Penicillium decumbens pTBG-1 transformants

[0059] Using the extracted transformant chromosomal DNA as a template and B1S / B3A as a primer pair (see Example 1) for PCR amplification. PCR reaction system (20μl): 10×EF Taq buffer 2.0μl; dNTP mix (10mmol / L) 0.4μl; B1S and B3A (10nmol / ml) 1μl each; EF-Taq (2.5U / μl) 0.1μl; template 1μl ; make up the volume to 20 μl with sterile water. The PCR cycle program was as follows: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 56.3°C for 30 sec, and 72°C for 70 sec; 72°C for 10 min.

[0060] The above PCR verification of Penicillium decumbens transformants (Gi24-2, Gi31-2, Gi53-2, Gi54-2, Gi55-1, Gi72-3, Gi78-2) found that all of them could amplify to the fragment size The bgl1 cDNA gene fragment of about 2.3kb is consistent with the size of the cDNA fragment of Trichoderma reesei bgl1, but no corresponding band appears in the starting strain, and the results are as follows figure 2 , indicating that...

Embodiment 3

[0061] Example 3. Southern blot verification of Penicillium decumbens pTBG-1 transformants

[0062] Southern blot analysis of bgl1 copy number of transformants. Using B4S / B3A as a primer pair (wherein, B3A refers to Example 1, and B4S is GAGCAACCCAGATGACCG), Trichoderma reesei chromosomal DNA is used as a template for PCR amplification, and the obtained gene fragments are purified and labeled with a gel recovery kit as probes (See DIG High Primer DNALabeling and Detection Starter Kit I, Roche's instructions for details). The PCR reaction program was: 94°C for 5min; 30 cycles of 94°C for 30s, 59°C for 30s, and 72°C for 53s; 72°C for 10min. The amplified fragment is 1688bp. Inoculate the starting strain and Gi series Penicillium decumbens transformants into 50ml basic medium respectively, culture at 200rpm 30°C for 2 days, filter the mycelia, extract a large number of chromosomes, and digest the extracted chromosomes with restriction endonuclease XhoI Overnight, 30V electroph...

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Abstract

The invention discloses a penicillium deumbens engineered strain containing over-expressed beta-glucosidase, which contains over-expressed beta-glucosidase genes, is named penicillium deumbens Gi31-2 and is preserved in China Center for Type Culture Collection on May 7, 2010 with a preservation number of CCTCC M 2010111. The invention also discloses the application of the strain in production of the beta-glucosidase and degradation of cellulose. The highest activity of the beta-glucosidase of the engineered strain of the invention reaches 3.47 IU / ml and is 2.90 times as high as that of an original host strain, while highest filter paper activity is 3.32 IU / ml and is 1.23 times as high as that of the original host strain. Therefore, the strain is widely applied to the production of the beta-glucosidase.

Description

technical field [0001] The invention belongs to the field of microbial engineering; specifically, the invention relates to a strain of Penicillium decumbens engineering bacteria overexpressing β-glucosidase, and its application in producing β-glucosidase and degrading cellulose. Background technique [0002] β-glucosidase (β-glucosidase, E.C.3.2.1.21), also known as cellobiase, is an important enzyme component in the process of cellulose degradation. β-glucosidase plays a key role in saccharification and hydrolysis of cellulose. It can degrade cellobiose, relieve the feedback inhibition effect of excessive cellobiose accumulation on cellobiohydrolase and endoglucanase, and improve the whole fiber Efficiency of the enzyme system to degrade cellulosic substrates. However, the analysis of the cellulase system components of cellulase-producing filamentous fungi shows that the proportion of β-glucosidase in the cellulase system is very low, and this low content of β-glucosidase ...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N9/42C12R1/80
Inventor 汪天虹杜春梅钟耀华李忠海张艳美
Owner SHANDONG UNIV
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