Low temperature alkaline pectinase mutant with improved specific activity and thermal stability

A technology of thermal stability and mutant, applied in the field of bioengineering, can solve problems such as poor thermal stability and unfavorable industrial application, and achieve the effects of improving thermal stability, activity, specific enzyme activity and thermal stability.

Active Publication Date: 2018-09-28
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The alkaline pectinase gene studied by the inventor in the previous period comes from the patent 201710497480.0, which contains an open reading frame of 993bp and encodes 331 amino acids. The low-temperature alkaline pectin lyase obtained by expressing the gene pel1 in Escherichia coli BL21(DE3) The study of enzymatic property analysis after purification shows that the optimum temperature of the enzyme is 30°C, the optimum pH is 10

Method used

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  • Low temperature alkaline pectinase mutant with improved specific activity and thermal stability
  • Low temperature alkaline pectinase mutant with improved specific activity and thermal stability
  • Low temperature alkaline pectinase mutant with improved specific activity and thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, Obtaining of Low Temperature Alkaline Pectin Lyase Mutant E184D / K185S

[0026] According to the nucleotide sequence of the wild-type low-temperature alkaline pectin lyase gene pel1, the point mutation primer pair is designed as follows:

[0027] E184D / K185S-F: 5'CAGC GATAGCG ACACCCTCAAT 3'

[0028] E184D / K185S-R: 5'GTGTC GCTATC GCTGTAACCATTGA 3'

[0029] Using the plasmid pET26a-pel 1 as a template, PCR amplification was performed with the designed primer pair.

[0030] PCR reaction system:

[0031]

[0032] PCR reaction conditions: pre-denaturation at 98°C for 30s, then denaturation at 98°C for 10s, annealing at 55°C for 5s, extension at 72°C for 30s, 25 cycles, and finally extension at 72°C for 30s.

[0033]The yield and specificity of the PCR products were detected by 0.7% agarose gel electrophoresis, and purified with a DNA purification kit. The purified PCR product was demethylated with DpnI, transformed into Escherichia coli XL-Gold clone com...

Embodiment 2

[0034] Embodiment 2, expression and purification of low temperature alkaline pectin lyase

[0035] Plasmids pET26b-pel1 and pET26b-pelE184D / K185S were respectively transferred into BL21(DE3) to obtain recombinant bacteria BL21(DE3) / pET26b-pel1 and BL21(DE3) / pET26b-pelE184D / K185S.

[0036] The two recombinant bacteria BL21(DE3) / pET26b-pel1 and BL21(DE3) / pET26b-pelE184D / K185S were respectively cultured in TB medium containing 50 μg / ml kanamycin, and cultured at 37°C for 3 hours; OD 600 When =2.0-2.5, add IPTG to its final concentration in LB medium of 0.5mM, transfer to 18°C ​​and continue culturing for 25-30h.

[0037] Collect the fermentation supernatant by centrifugation at 5000rpm and 10min, desalt the supernatant with a desalting column GE HiTrap Desalting, and elute with solution C (50mM Tris-HCl, pH 7.0), and then pass the eluate through an anion exchange column GEHiTrap SP FF, first use solution D (20mM Tris-HCl, pH7.5) to elute the impurity protein, then use solution E...

Embodiment 3

[0039] Embodiment 3, the comparison of the specific enzyme activity of low temperature alkaline pectin lyase Pel1 and E184D / K185S

[0040] 1. Determination of pectinase activity

[0041] Preheat the PGA solution at 30°C for 5 minutes; take 20 μl of enzyme diluent, add 2ml of buffer solution containing 0.2% PGA to start the enzymatic reaction; the reaction conditions are 30°C, 15min, and then stop the reaction with 3ml of 0.03mol / L phosphoric acid. The absorbance value was measured at 235nm.

[0042] Blank control: inactive enzyme solution reaction (namely: first take 3ml of phosphoric acid and mix with the enzyme solution to be tested, react according to the above reaction conditions, and then add the substrate to terminate the reaction).

[0043] Enzyme activity calculation:

[0044]

[0045] Where: 4600(L . mol -1 cm -1 )—the molar absorptivity of unsaturated PGA at 235nm;

[0046] t (min)—enzymatic reaction time (in the linear range of enzyme reaction);

[0047] b...

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Abstract

The invention discloses a low temperature alkaline pectinase mutant with the improved specific activity and thermal stability. The mutant has an amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence with low temperature alkaline pectin lyase activity obtained by deletion, replacement, insertion or/and addition of conserved mutations of one to several amino acids on the basis of the amino acid sequence shown in SEQ ID NO.1. The mutant can greatly improve the specific activity and thermal stability, and is more suitable for industrial production in the fields such as textile degumming and washing and meets the needs of social production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a low-temperature alkaline pectin lyase mutant with improved specific enzyme activity and thermal stability. Background technique [0002] Pectinase refers to the general term for various enzymes that can catalyze the decomposition of pectin. Pectinase was originally extracted from oranges by MacDonnell. Pectin—the substrate of pectinase widely exists in higher plants, and is an important component of plant interstitium and primary cell wall, and plays a role of "glue" in plant cell tissues. The research on pectin structure chemistry shows that the chemical structure of pectin is relatively complex, and there are many kinds of pectinases that can catalyze its decomposition. Pectinases can be divided into three categories according to the reaction properties of decomposing glycosidic bonds or the properties of degrading substrates: pectin hydrolases, pectin lyases and pec...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21
CPCC12N9/88C12Y402/02002
Inventor 张桂敏唐雨蒙巫攀易犁马延和
Owner HUBEI UNIV
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