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Preparation and application of sclerotium gum hydrolase mutant

A technology of sclerotiorum gum and hydrolase, which is applied in the field of enzyme engineering and can solve the problems of limited hydrolysis of sclerotium sclerotiorum, inability to meet industrial application requirements and the like

Active Publication Date: 2021-04-16
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The inventor's team screened an endogenous sclerotium hydrolase from Sclerotium rolfsii in the previous research work (recorded in the patent application document with application number 202011172161.0), which has the ability to hydrolyze sclerotium ability, but the enzyme activity cannot meet the needs of industrial applications, and the hydrolysis of sclerotin is limited. Therefore, it is necessary to further strengthen the activity of hydrolytic enzymes and reduce the molecular weight of sclerotin, thereby expanding its industrial application range

Method used

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  • Preparation and application of sclerotium gum hydrolase mutant
  • Preparation and application of sclerotium gum hydrolase mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Expression of wild-type Sclerotinia hydrolase

[0037] After the Pichia pastoris (the patent application document with the publication number of CN112266907A) that was constructed and preserved in the laboratory to express the Sclerotinia hydrolase (GME9860) gene was activated on the YPD plate, a single colony was picked and inoculated in 2 mL of YPD medium. In a test tube, cultured at 30°C, 220r / min for 24h, transferred to a 250mL shake flask containing 50mL of BMGY medium with 2% inoculum, and cultivated to OD at 30°C, 220r / min 600 = 2-6, collect the bacteria by centrifugation, wash twice with normal saline, put them all into a 250mL shake flask containing 50mL of BMMY medium, culture at 30°C and 220r / min, add methanol to the medium every 24h until the final The concentration is 0.5%. After 120 hours of cultivation, the bacterial cells are removed by centrifugation, and the supernatant is the crude enzyme solution. After testing, the enzyme activity of the...

Embodiment 2

[0038] Example 2: Preparation and expression of a single mutant of Sclerotinia hydrolase

[0039] (1) Construction of sclerotinia hydrolase mutant

[0040] Using rapid PCR technology, according to the gene sequence (SEQID NO.1) of the Sclerotium rolfsii Sclerotium sclerotin hydrolase, respectively design and synthesize primers that introduce mutations of D36E, A76V, D144E, T161Y, S180D to Sclerotium sclerotin hydrolase Genes were subjected to site-directed mutation (underlined bases are mutated bases).

[0041] The site-directed mutagenesis primers for introducing the sequence D36E mutation are:

[0042] Forward primer: 5'-CGACTACGTGG GAG TGCTGCGAACCT-3'

[0043] Reverse primer: 5'-AGGTTCGCAGCACTCCCACGAGTCG-3'

[0044] The site-directed mutagenesis primers for introducing sequences such as A76V mutation are:

[0045] Forward primer: 5'-CAAAATGCCTGCTTT GTT AGCGGCGGAAAT-3'

[0046] Reverse primer: 5'-ATTTCCGCCGCTAACAAAGCAGGCATTT-3'

[0047] The site-directed mutagenesis...

Embodiment 3

[0061] Embodiment 3: the performance analysis of Sclerotinia pectin hydrolase

[0062] Detection of hydrolysis ability of sclerotinia: 1mL reaction solution containing 500 μL of crude enzyme solution and 500 μL of sclerotinia, inactivated in 30°C water bath for 6 hours, 100°C in boiling water bath for 5 minutes, using inactivated enzyme as a control, using high performance liquid chromatography ( HPGPC) to determine the molecular weight of polysaccharides. Utilize Shodex SB-806M HQ gel chromatographic column detection, detection conditions are: differential detector, mobile phase 0.1mol / L NaNO 3 Solution, flow rate 0.6mL / min, column temperature 40°C, injection volume 50μL.

[0063] The wild-type sclerotinia hydrolase (WT) and mutant shake flask cultured 120h crude enzyme activity and the ability to hydrolyze sclerotinia are listed in Table 1. The results show that the enzyme activity of each mutant is higher than that of the wild type , the ability to hydrolyze sclerotin inc...

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Abstract

The invention discloses preparation and application of a sclerotium gum hydrolase mutant, and belongs to the technical field of gene engineering and enzyme engineering. The invention provides a series of sclerotium gum hydrolase mutants. The sclerotium gum hydrolase activities of the mutants D36E, A76V, D144E, T161Y, S180D, D36E / D144E, D36E / S180D, D144E / S180D, D36E / D144E / S180D are respectively improved by 20.1%, 14.2%, 22.2%, 15.6%, 18.3%, 51.4%, 63.0%, 48.6% and 92.9% compared with the wild type, and the corresponding sclerotium gum molecular weights are respectively reduced by 42.7%, 30.7%, 47.2%, 32.9%, 48.2%, 55.2%, 59.4%, 57.4% and 72.9% compared with the wild type. The reduction of the molecular weight of sclerotium gum is more beneficial to widening the industrial application range of sclerotium gum.

Description

technical field [0001] The invention relates to the preparation and application of a sclerotinia hydrolase mutant and belongs to the technical field of enzyme engineering. Background technique [0002] Sclerotin (also known as scleroglucan, scleroglucan) is a non-ionic water-soluble microbial exopolysaccharide composed of β-1,3-glucopyranose and β-1,6-glucopyranose residues On average, every 3 main chain units linked by β-1,3 glycosidic bonds are connected with one side chain of β-1,6 glycosidic bonds. This repeating unit structure makes the branching degree of sclerotinium reach about 0.33. In addition to the biological activity characteristics shared by most β-1,3 glucans, Sclerotin has high water solubility due to its high branching frequency. Due to its unique chemical structure and high molecular weight properties, Sclerotinium has good water solubility, pseudoplasticity, moisture retention, salt resistance and viscosity stability, and is widely used in food, biomedic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12R1/84
Inventor 曾伟主周景文单小玉陈坚堵国成
Owner JIANGNAN UNIV
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