CN-121991939-A - Alkaline pectate lyase PL1B and encoding gene and application thereof

Abstract

The invention discloses an alkaline pectate lyase PL1B, and a coding gene and application thereof, wherein the amino acid sequence of the alkaline pectate lyase PL1B is shown as SEQ ID NO.1, and the nucleotide sequence of the coding alkaline pectate lyase PL1B gene is shown as SEQ ID NO. 2. The invention successfully realizes the heterologous expression of pectate lyase PL1B by a genetic engineering means, and obtains alkaline pectate lyase PL1B after purifying the heterologously expressed pectate lyase PL1B by Ni-NTA agarose purification resin. Experimental results show that the optimal temperature of the alkaline pectate lyase PL1B is 35 ℃ and the optimal pH value is 8.5 under the condition of no exogenous Ca 2+ , and the alkaline pectate lyase PL1B has degradation activity on PGA pectin, citrus pectin and polygalacturonic acid (PGA). Therefore, the pectate lyase PL1B can be widely applied to the fields of agriculture, industry, food additives, medicine, production of pectin oligosaccharide with prebiotic effect and the like, and has good application prospect.

Inventors

• Yan fen
• LU HAN
• Hong Jianqu
• NIU XIAOXU
• REN XIAOMIN

Assignees

• 福州大学

Dates

Publication Date

20260508

Application Date

20260120

Claims (6)

1. 1. The basic pectate lyase PL1B is characterized in that the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence of the basic pectate lyase PL1B gene is shown as SEQ ID NO. 2.
2. 2. A process for preparing the alkaline pectate lyase PL1B as claimed in claim 1, comprising the steps of: s1, cloning a gene for encoding alkaline pectate lyase PL1B into a pET28a plasmid to obtain a recombinant expression vector pET28a-PL1B; S2, transforming a recombinant expression vector pET28a-PL1B into host bacteria E.coil BL21 (DE 3) to obtain genetically engineered bacteria E.coil BL21 (DE 3) -pET28a-PL1B; S3, performing IPTG induction culture on the genetically engineered bacterium E.coil BL21 (DE 3) -pET28a-PL1B, separating and purifying to obtain recombinant pectate lyase PL1B.
3. 3. The method for preparing the alkaline pectate lyase PL1B according to claim 2, wherein the condition of IPTG induction culture is that the final concentration of IPTG is 0.8mM, the induction culture temperature is 20 ℃, and the induction culture time is 16h.
4. 4. The method for producing an alkaline pectate lyase PL1B according to claim 2, wherein the purification is performed by a Ni-NTA column.
5. 5. Recombinant expression vector pET28a-PL1B comprising a gene encoding the alkaline pectate lyase PL1B of claim 1.
6. 6. Use of the alkaline pectate lyase PL1B according to claim 1 or of the gene encoding the alkaline pectate lyase PL1B in the field of production of pectic oligosaccharides with prebiotic effect in agriculture, industry, food additives, medicine.

Description

Alkaline pectate lyase PL1B and encoding gene and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to alkaline pectate lyase PL1B and a coding gene and application thereof. Background Pectin (Pectin) is an acidic macromolecular heteropolysaccharide with D-galacturonic acid (GalA) linked by alpha-1, 4-glycosidic bond, and is an important component of plant cell wall, and widely distributed in root, stem, leaf and fruit of higher plants. Pectin from different sources varies in the Degree of Esterification (DE). Fruit gums are classified according to the degree of esterification into high methoxy pectin (DE > 50%) and low methoxy pectin (DE < 50%). Pectin can be classified into homogalacturonic acid (HGA), rhamnogalacturonate I (RGI), rhamnogalacturonate II (RGII) and Xylogalacturonate (XGA) according to structural types. Wherein about 65% of the area of pectin belongs to HG and consists essentially of linear chains of alpha-1, 4-glycosidically linked galacturonic acid. The biological activity of pectin is generally manifested in that degradation products obtained by partial enzymatic hydrolysis have a broad range of prebiotic effects. Accordingly, pectin Oligosaccharides (POS), a degradation product of pectin, are of increasing interest. Pectin Oligosaccharide (POS) is also called as oligomeric galacturonic acid, which is an oligosaccharide product with relatively low molecular weight produced by depolymerizing pectin molecules, and is formed by linking alpha-1, 4-glycosidic bonds with galacturonic acid as a basic unit, wherein the polymerization degree is usually between 2 and 10, and the molecular weight is between 200 and 2000 Da. The esterification degree, solubility and gel characteristics of pectin oligosaccharides are different from those of pectin according to the structure and relative molecular mass of pectin after degradation, so that the pectin oligosaccharides have more important values in the aspects of industrial application, scientific research, promotion of human health and the like. POS can be selectively utilized by the gut microbiota for fermentation, and is considered the best choice for the second generation prebiotic factors. Numerous studies have shown that POS exhibit a variety of physiological activities, such as prebiotic, antibacterial, anticancer and antioxidant properties, and can be developed as a functional food additive. In addition, pectin oligosaccharides can be used in pharmaceuticals, cosmetics, animal feeds, and functional foods for patients suffering from obesity and diabetes. Compared with physical and chemical methods, the method for depolymerizing pectin by enzymatic reaction has the advantages of mild reaction conditions, high specificity, high degradation efficiency and the like. Thus, enzymatic processes for the preparation of pectin oligosaccharides with good biological activity are of increasing interest. Pectase is a generic term of a series of enzymatic hydrolysis pectic substances, and pectate lyase is the only enzyme class of pectate enzyme capable of degrading pectin with high esterification degree and generating no methanol, and can be divided into acid pectate lyase and alkaline pectate lyase according to the acid-base property of enzyme acting substrate environment. Acid pectate lyase has been widely used in the food industry for extraction and clarification of fruit juices and wines, and alkaline pectate enzymes have potential applications in plant fiber degumming, coffee fermentation and bioenergy. The microbial strain is a main producer of pectate lyase, and most microbial (wild) strains producing pectate lyase have the problems of low enzyme yield, low catalytic efficiency and the like, thereby limiting industrial large-scale application. Disclosure of Invention Aiming at the technical problems in the background technology, the invention aims to provide an alkaline pectate lyase PL1B and a coding gene and application thereof. In order to achieve the above purpose, the present invention adopts the following technical scheme: in a first aspect, the invention provides an alkaline pectate lyase PL1B derived from Paenibacillus, the amino acid sequence of which is shown in SEQ ID NO. 1. In a second aspect, the invention provides a gene PL1B encoding the above basic pectate lyase PL1B, the nucleotide sequence of which is shown in SEQ ID NO. 2. The third aspect of the present invention provides a method for preparing the alkaline pectate lyase PL1B derived from Paenibacillus, comprising the steps of: s1, cloning the gene for encoding the alkaline pectate lyase PL1B into a pET28a plasmid to obtain a recombinant expression vector pET28a-PL1B; S2, transforming a recombinant expression vector pET28a-PL1B into host bacteria E.coilBL21 (DE 3) to obtain genetically engineered bacteria E.coilBL21 (DE 3) -pET28a-PL1B; S3, performing IPTG induction culture on the genetically engineered bac