CN-122012480-A - Pectase, and preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to pectase and a preparation method and application thereof. The amino acid sequence of pectase is shown as SEQ ID NO. 1. The nucleotide sequence of the pectase encoding gene is shown as SEQ ID NO. 2. The invention also provides application of the pectase or the recombinant expression vector or the recombinant expression transformant thereof in degrading tobacco pectin, releasing aroma components and improving the aroma quality of tobacco. The pectase of the invention shows high-efficiency pectic degradation capability of tobacco, and the content of 20 aroma substances in the tobacco treated by the pectase is obviously increased, in particular to compounds such as solanone, 4,7, 9-megastigmatrien-3-one, dihydro-kiwi lactone, and lotione, and the like, which have obvious aroma enhancement effect on smoke.

Inventors

• WANG ZHEN
• CHEN YUNFEI
• WU NAN
• ZHENG SHUJUAN
• WEI JINBIN
• YANG HONGJING
• WANG ZHICAI
• SONG KAI
• ZANG ZHIPENG
• GAO YUZHEN

Assignees

• 甘肃烟草工业有限责任公司

Dates

Publication Date

20260512

Application Date

20260205

Claims (10)

1. 1. The pectase is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. 2. A pectinase encoding gene encoding the pectinase of claim 1.
3. 3. The pectase encoding gene according to claim 2, wherein the nucleotide sequence of the pectase encoding gene is shown in SEQ ID NO. 2.
4. 4. A recombinant expression vector comprising a vector and a pectinase encoding gene according to any one of claims 2 to 3.
5. 5. The recombinant expression vector according to claim 4, wherein said vector is pET-28a (+).
6. 6. A recombinant expression transformant comprising the recombinant expression vector of any one of claims 4 to 5.
7. 7. The use of the pectinase of claim 1, the pectinase encoding gene of any one of claims 2 to 3, the recombinant expression vector of any one of claims 4 to 5 and the recombinant expression transformant of claim 6, wherein the pectinase of claim 1, the pectinase encoding gene of any one of claims 2 to 3, the recombinant expression vector of any one of claims 4 to 5 or the recombinant expression transformant of claim 6 is used for degrading tobacco pectin, releasing aroma components and improving the aroma quality of tobacco.
8. 8. A method for preparing pectase, which is characterized by comprising the following steps: (1) Introducing pectase encoding gene into a vector plasmid pET-28a (+) with a histidine tag to obtain a recombinant expression vector, and transforming the recombinant expression vector into competent cells of escherichia coli BL21 (DE 3) to obtain a recombinant expression transformant; (2) Inoculating recombinant expression transformant into LB liquid medium containing kanamycin, shake culturing overnight, transferring to fresh LB medium, culturing until OD600 reaches 0.8, and adding IPTG to induce expression; (3) After the induction is finished, centrifugally collecting thalli, washing the thalli with sterile water, centrifugally taking out sediment, re-suspending the thalli and a lysis buffer solution, crushing in an ice bath, and purifying the crushed crude enzyme solution to obtain pectase.
9. 9. A method of flavoring tobacco comprising the steps of: (1) Mixing pectase and tobacco according to a mass ratio of 1:2000-1:50; (2) Static treatment is carried out for 1 to 10 hours at the temperature of 30 to 55 ℃; (3) Inactivating at 50-200deg.C for 1-60 min to obtain flavored tobacco.
10. 10. A method of flavoring tobacco as claimed in claim 9, wherein, In the step (1), pectase and tobacco are mixed according to the mass ratio of 1:1000; in the step (2), static treatment is carried out for 6 hours at the temperature of 40 ℃; in step (3), the inactivation is carried out for 1-2 minutes at 160 ℃.

Description

Pectase, and preparation method and application thereof Technical Field The invention relates to the technical field of bioengineering, in particular to pectase and a preparation method and application thereof. Background Pectin is a key adhesive component of tobacco cell interstitium and has a core effect on maintaining the tissue structural integrity and mechanical toughness of tobacco leaves. Proper amount of pectin can ensure the structural stability of tobacco leaves and improve the moisture absorption performance of the tobacco leaves through osmosis regulation. However, when the pectin content in the tobacco is too high, not only the cigarette is not burnt sufficiently, but also small molecular substances such as methanol generated by pyrolysis of the cigarette can obviously reduce the smoking quality of the tobacco. Meanwhile, in the natural aging process of tobacco leaves, pectin can be gradually converted into acetic acid, so that uncomfortable reactions such as cough, throat pricking and the like of smokers are caused, a stimulating peculiar smell is generated, the purity and sensory comfort of smoke are seriously damaged, and in addition, the pectin can be decomposed to generate various volatile harmful components in the combustion stage, so that potential threat is formed to human health. In addition, although partial pectin can be degraded after the tobacco leaves are treated by the primary baking and redrying processes, a large amount of pectin is still difficult to be further decomposed due to structural changes such as molecular chain cross-linking polymerization, esterification group removal and the like. This phenomenon can lead to stiffness in the texture of the tobacco after modulation, elastic attenuation and reduction of the breakage resistance, and meanwhile, the filling value is obviously reduced, so that the processing applicability of the tobacco is seriously affected. Pectase is a generic term for a class of enzymes that break down pectin, and mainly includes protopectase, polygalacturonase, pectin lyase, and pectin esterase. Among them, oligogalacturonase (Oligogalacturonate lyase, OGL) is also called pectin lyase (PENTINLYASES, PL), belonging to both the pectin lyase family and the polysaccharide lyase (Polysaccharide Lyase, PL) family. OGL acts mainly on the later stages of pectin degradation, catalyzing the degradation of de-esterified pectin or oligogalacturonan in an exo-type manner. The beta-trans elimination effect specifically cleaves the alpha-1, 4-glycosidic bond of the oligogalacturonic acid to generate unsaturated oligogalacturonic acid, thereby participating in the degradation metabolism of pectin polysaccharide. It is worth noting that the processed tobacco pectin usually forms a special stable conformation, and conventional microbial pectase and commercial pectase preparations often have problems of mismatched substrate specificity, insufficient catalytic efficiency, incapability of effectively penetrating to a substrate action site and the like, so that efficient degradation of the tobacco pectin is difficult to realize. Aiming at the complex structural characteristics and transformation rules of tobacco pectin, the special pectase with high specificity and strong adaptability is developed. The preparation becomes a key technical break for accurately regulating and controlling the physical properties of tobacco leaves and improving the sensory quality. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide pectase, a preparation method and application thereof, and solves the problem of low degradation efficiency of tobacco pectin. In a first aspect of the invention, a pectase is provided, the amino acid sequence of which is shown as SEQ ID NO. 1. In a second aspect of the invention, there is provided a pectinase encoding gene encoding the pectinase. Preferably, the nucleotide sequence of the pectase encoding gene is shown as SEQ ID NO. 2. In a third aspect of the invention, there is provided a recombinant expression vector comprising a vector and said pectinase encoding gene. Specifically, the pectase encoding gene of the present invention is constructed by ligating it to various suitable vectors by a method conventional in the art. The vector can be various conventional vectors in the field, the expression vector is selected from plasmids, and the plasmid is selected from pET series expression plasmids, preferably pET-28a (+). The pectinase encoding gene may be operably linked downstream of regulatory sequences suitable for expression in the selected vector to effect constitutive or inducible expression of the pectinase. Preferably, the recombinant expression vector is kanamycin or tetracycline resistant. In a fourth aspect of the present invention, there is provided a recombinant expression transformant comprising the recombinant expression vector. Specifically, the recombinant expression transformant is prod