Lentinus edodes laccase LeLac11 and application thereof in improvement of stress tolerance of microorganisms

A microbial and stress-resistant technology, applied in the field of oxidase, can solve problems such as quality decline, mycelium growth rate decline, and shiitake mushroom production reduction

Active Publication Date: 2021-05-11
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lentinus edodes belongs to wood-rot fungi, and its cultivation and production require the degradation of lignin to provide sufficient nutrients for mycelial growth and fruiting body development, and it is a medium-low temperature type of edible fungus, and the growth rate of mycelium decreases signifi...

Method used

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  • Lentinus edodes laccase LeLac11 and application thereof in improvement of stress tolerance of microorganisms
  • Lentinus edodes laccase LeLac11 and application thereof in improvement of stress tolerance of microorganisms
  • Lentinus edodes laccase LeLac11 and application thereof in improvement of stress tolerance of microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construction of Laccase LeLac11 Overexpression Vector pET30a / LeLac11 in Example 1 Lentinus edodes 18

[0050] First, design primers to amplify the target gene LeLac11 fragment from the original plasmid, and then recombine it into the digested overexpression vector pET30a through the seamless cloning recognition sites at both ends of the primer; transfer the ligated product into the prepared The competent cells of Escherichia coli BL21 were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing and identification of the grown monoclonal colonies. The correct clone sequence was compared to the successfully constructed overexpression plasmid pET30a / LeLac11. After success, the graph is as follows Figure 5 shown.

[0051] 1. Design and synthesize primers

[0052] (1) Design PCR amplification fragment primers, and introduce homologous sequences at the ends of the linearized cloning vector at the 5' end of the primers, so that the 5' and 3' end sequences of the a...

Embodiment 2

[0088] Prokaryotic expression of embodiment 2 mushroom laccase LeLac11 gene

[0089] 1. Inoculate a single colony of Escherichia coli BL21 containing pET30a / LeLac11 in 100mL LB liquid medium containing 100μg / mL Kan, culture at 220rpm, 37℃ until the OD600 reaches 0.6-0.8, and then add IPTG with a concentration of 1mM to In 100mL liquid LB, continue to culture at 220rpm, 37°C for 4h, in order to achieve the purpose of optimizing the induction culture conditions. At the same time, the empty vector pET30a was transformed into BL21 as a control.

[0090]2. Extract prokaryotic expressed protein for SDS-PAGE electrophoresis

[0091] ①Protein sample preparation

[0092] (1) Take 1 mL of the bacterial solution from Step 1 and add it to 100 mL of LB liquid medium containing 100 μg / mL Kan, incubate at 37°C and 220 r / min for 2.5-3 hours, and use a UV spectrophotometer to detect when the OD 600 reaches 0.4-0.6 , adding IPTG with a concentration of 1 mM for induction;

[0093] (2) After...

Embodiment 3

[0107] Embodiment 3 Research on the cold-resistant function of Lentinus edodes laccase LeLac11

[0108] 1. Take 50 μL of the verified bacterial solution, inoculate it into 50 mL of LB (containing 100 μg / mL Kan) liquid medium, incubate at 37°C and 150 r / min for 2.5-3 hours, and use a UV spectrophotometer to detect when the OD600 reaches 0.4-0.6 , adding 1 mM IPTG for induction.

[0109] 2. Placed in an incubator at 4°C, and subjected to cold stress for 12h, 24h, 36h, 48h, 60h.

[0110] 3. Take 3 mL of the bacterial liquid after cold stress treatment, detect the absorbance at 600 nm with an ultraviolet spectrophotometer, and record the data. Three replicates were set for each experimental gradient.

[0111] 4. Take the absorbance of the OD600 of the bacterium solution at 0h as a control, calculate the growth rate of Escherichia coli containing pET30a / LeLac11 and Escherichia coli containing pET30a, and make a line graph of growth rate after cold stress (note: growth rate=cold-t...

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Abstract

The invention discloses lentinus edodes laccase LeLac11 and application thereof in improvement of stress tolerance of microorganisms. The amino acid sequence of the lentinus edodes laccase LeLac11 is shown as SEQ ID NO.1, and the nucleotide sequence of a coding gene of the lentinus edodes laccase LeLac11 is shown as SEQ ID NO.2. The lentinus edodes laccase LeLac11 gene is transferred into a suitable microbial host, so that the host expresses the laccase, and the cold stress resistance, heat stress resistance, salt stress resistance and heavy metal lead and cadmium stress resistance of host cells are improved. The lentinus edodes laccase LeLac11 is derived from fungi, and the stress tolerance of the host can be improved by transferring the lentinus edodes laccase LeLac11 into the suitable microbial host.

Description

technical field [0001] The invention belongs to the technical field of oxidases, and relates to a mushroom laccase LeLac11 and its application in improving the stress resistance ability of microorganisms. Background technique [0002] Lentinula edodes (Berk.) Pegler belongs to Basidiomycota, Agaricaceae, Agaricales, Phytophthora, and Lentinula genus in taxonomy. Lentinus edodes belongs to wood-rot fungi, and its cultivation and production require the degradation of lignin to provide sufficient nutrition for mycelial growth and fruiting body development, and it is a medium-low temperature edible fungus, and the growth rate of mycelium decreases significantly at 30 °C (Wang Bo et al. Experimental study on the growth of mycelium and fruiting bodies of Lentinus edodes strains against high temperature [J]. Journal of Jilin Agricultural University, 2004, 26(2): 145-147), high temperature directly leads to the reduction of yield and quality of Lentinus edodes. [0003] Laccase (La...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/80C12N1/21C12N1/15C12R1/19
CPCC12N9/0061C12N15/70C12N15/80C12Y110/03002
Inventor 赵妍陈明杰杨焕玲仝宗军隽加香董沁李治平王耀冉
Owner SHANGHAI ACAD OF AGRI SCI
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