Method for improving optimum temperature of family-10 xylanase

Abstract

The invention aims to provide a high-efficiency expression and purification method for family-10 xylanase Aus Xyn10A thermostability modified and reconstructed mutant enzyme. According to a comparison result of protein sequences of the Aus Xyn10A (derived from Aspergillus usamii E001) and thermostable xylanase (derived from Thermotoga maritima), the C terminal repeat of thermostable enzyme is used to the C terminal of the Aus Xyn10A by a gene engineering method, and the obtained mutant enzyme is named Aus Xyn10A'. Experimental results prove that the optimum temperature of the mutant enzyme is obviously improved, and the thermostable enzyme serving as a thermostable enzyme preparation has high industrial production potential and economic value.

Description

technical field

[0001] The invention relates to a high-efficiency expression method for transforming xylanase of the 10th family and a mutant enzyme, and belongs to the technical field of bioengineering. Background technique

[0002] With the continuous growth of population and further consumption of resources, the development and utilization of renewable resources has become a common concern of the world. Xylan is an important component of hemicellulose, and its content in plant cell walls is second only to cellulose, accounting for about one-third of the dry weight of cells. Xylan is a hybrid polymer molecule, the main chain is connected by multiple xylopyranosyl groups through xylosidic bonds. According to the type of glycosidic bonds, xylan can be divided into two types, namely β-1,4-xylan and β-1,3-xylan. The former is found in the cell walls of terrestrial plants, while the latter is mainly found in the cell walls of marine algae. Depending on the source of the stra...

Examples

Embodiment 1

[0019] The construction of embodiment 1 mutant enzyme

[0020] Extract the T vector (pUCm-T-Aus xyn10A) containing the Aus Xyn10A gene, and use the mature peptide primer Xyn10A-F1 (5'-GAATTCCAGGCTTCAGTGAGTATTGA-3') of the Aus Xyn10A gene as a template to carry out the first round of PCR, The reaction conditions are: 94°C for 5 minutes; 2 cycles of 94°C for 30s, 45°C for 30s, and 72°C for 70s; 28 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 70s; 72°C for 10 minutes; Using the first-round PCR product as a template, the second-round PCR was carried out using primers Xyn10A-F1 and primer XynCHR2. The reaction conditions were: 94°C for 5 minutes; 2 cycles, 94°C for 30s, 45°C for 30s, and 72°C for 70s; 28 cycles Cycle 94°C for 30s, 55°C for 30s, 72°C for 70s; 72°C for 10min; store at 10°C. The two rounds of PCR amplification products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by cutting the gel and ligated with the pUCm-T vector (pUCm-T-Aus...

Embodiment 2

[0021] Example 2 Containing the Construction of the Expression Plasmid Encoding Aus Xyn10A' Mature Peptide Gene

[0022] Use EcoR I and Not I to double-enzyme digest the target gene recovered from rubber tapping and pPIC9K respectively. The digestion time is 4 hours. The digested product recovered from rubber tapping is ligated overnight (>12 hours) under the action of T4 DNA ligase to obtain the recombinant plasmid pPIC9K -Ausxyn10A'( figure 1 ), and sequenced the recombinant expression plasmid.

Embodiment 3

[0023] Example 3 Construction, expression, product purification and activity determination of GS115 / Aus xyn10A'

[0024] Linearize pPIC9K-Aus xyn10A' with Sal I, perform electrotransformation and screening according to the Pichia expression manual, and obtain high-copy Pichia recombinant GS115 / Aus xyn10A'. The engineered bacterium was induced with 0.5% methanol for 72 hours, and the recombinant xylanase activity in the fermentation broth was measured by DNS method up to 24IU / mL. The centrifuged supernatant is the recombinant xylanase crude enzyme solution, which is concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10kDa, and then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. After purification, It was detected as a single band by SDS-PAGE, and showed that the molecular weight of the recombinant xylanase was 42kDa. The optimum action temperature of the recombinant xylanase is 50°C,...

technical field

[0001] The invention relates to a high-efficiency expression method for transforming xylanase of the 10th family and a mutant enzyme, and belongs to the technical field of bioengineering. Background technique

[0002] With the continuous growth of population and further consumption of resources, the development and utilization of renewable resources has become a common concern of the world. Xylan is an important component of hemicellulose, and its content in plant cell walls is second only to cellulose, accounting for about one-third of the dry weight of cells. Xylan is a hybrid polymer molecule, the main chain is connected by multiple xylopyranosyl groups through xylosidic bonds. According to the type of glycosidic bonds, xylan can be divided into two types, namely β-1,4-xylan and β-1,3-xylan. The former is found in the cell walls of terrestrial plants, while the latter is mainly found in the cell walls of marine algae. Depending on the source of the stra...

Examples

Embodiment 1

[0019] The construction of embodiment 1 mutant enzyme

[0020] Extract the T vector (pUCm-T-Aus xyn10A) containing the Aus Xyn10A gene, and use the mature peptide primer Xyn10A-F1 (5'-GAATTCCAGGCTTCAGTGAGTATTGA-3') of the Aus Xyn10A gene as a template to carry out the first round of PCR, The reaction conditions are: 94°C for 5 minutes; 2 cycles of 94°C for 30s, 45°C for 30s, and 72°C for 70s; 28 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 70s; 72°C for 10 minutes; Using the first-round PCR product as a template, the second-round PCR was carried out using primers Xyn10A-F1 and primer XynCHR2. The reaction conditions were: 94°C for 5 minutes; 2 cycles, 94°C for 30s, 45°C for 30s, and 72°C for 70s; 28 cycles Cycle 94°C for 30s, 55°C for 30s, 72°C for 70s; 72°C for 10min; store at 10°C. The two rounds of PCR amplification products were analyzed by 1% agarose gel electrophoresis, the target band was recovered by cutting the gel and ligated with the pUCm-T vector (pUCm-T-Aus...

Embodiment 2

[0021] Example 2 Containing the Construction of the Expression Plasmid Encoding Aus Xyn10A' Mature Peptide Gene

[0022] Use EcoR I and Not I to double-enzyme digest the target gene recovered from rubber tapping and pPIC9K respectively. The digestion time is 4 hours. The digested product recovered from rubber tapping is ligated overnight (>12 hours) under the action of T4 DNA ligase to obtain the recombinant plasmid pPIC9K -Ausxyn10A'( figure 1 ), and sequenced the recombinant expression plasmid.

Embodiment 3

[0023] Example 3 Construction, expression, product purification and activity determination of GS115 / Aus xyn10A'

[0024] Linearize pPIC9K-Aus xyn10A' with Sal I, perform electrotransformation and screening according to the Pichia expression manual, and obtain high-copy Pichia recombinant GS115 / Aus xyn10A'. The engineered bacterium was induced with 0.5% methanol for 72 hours, and the recombinant xylanase activity in the fermentation broth was measured by DNS method up to 24IU / mL. The centrifuged supernatant is the recombinant xylanase crude enzyme solution, which is concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10kDa, and then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. After purification, It was detected as a single band by SDS-PAGE, and showed that the molecular weight of the recombinant xylanase was 42kDa. The optimum action temperature of the recombinant xylanase is 50°C,...

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