阅读说明：本技术 一种植物根系及果实促进生长用蛋白酶生产方法 (production method of protease for promoting growth of plant roots and fruits ) 是由 秦春国 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种植物根系及果实促进生长用蛋白酶生产方法,包括如下步骤：S1：相同或同属植物茎叶收集,选取相同可植物或同属科植物的茎叶,块茎,块根进行收集；S2：预处理,对收集到的茎叶,块茎或块根进行洗净,粉碎处理；S3：加入磷酸缓冲液,搅拌均浆,对粉碎物中加入磷酸缓冲液,充分搅拌均匀成浆；S4：过滤离心获得上清液和残渣,对匀浆进行过滤,并对过滤离心处理,获得上清液和残渣；S5：上清液硫酸铵盐析,离心收集上层不溶物,对上清液中加入硫酸铵盐析；本发明中,选取相同可植物或同属科植物的茎叶,块茎,块根进行收集,来生产蛋白酶,可以大大提升生产出的成品对植物根系及果实的促进生长作用,而且适应性更好,副作用更小。(the invention discloses a method for producing protease for promoting growth of plant roots and fruits, which comprises the following steps: s1: collecting stems and leaves of plants of the same or same genus, and collecting stems, leaves, tubers and root tubers of plants of the same or same genus; s2: pre-treating, namely cleaning and crushing the collected stem leaves, tubers or root tubers; s3: adding phosphate buffer solution, stirring and homogenizing, adding phosphate buffer solution into the crushed material, and fully and uniformly stirring to form slurry; s4: filtering and centrifuging to obtain supernatant and residue, filtering the homogenate, and centrifuging the filtration to obtain supernatant and residue; s5: salting out the supernatant by ammonium sulfate, centrifuging to collect insoluble substances on the upper layer, and adding ammonium sulfate into the supernatant for salting out; in the invention, the stem leaves, tubers and root tubers of the same plants or plants in the same family are selected for collection to produce the protease, so that the growth promoting effect of the produced finished products on plant roots and fruits can be greatly improved, the adaptability is better, and the side effect is less.)

1. a method for producing protease for promoting growth of plant roots and fruits is characterized in that: the method comprises the following steps:

s1: collecting stems and leaves of the same or same genus plant;

S2: pre-treating, namely cleaning and crushing the collected stem leaves, tubers or root tubers;

s3: adding phosphate buffer solution, stirring and homogenizing, adding phosphate buffer solution into the crushed material, and fully and uniformly stirring to form slurry;

s4: filtering and centrifuging to obtain supernatant and residue, filtering the homogenate, and centrifuging the filtration to obtain supernatant and residue;

s5: salting out supernatant with ammonium sulfate, centrifuging to collect upper insoluble substance, adding ammonium sulfate into supernatant, salting out, centrifuging, and collecting upper insoluble substance;

S6: dissolving the upper-layer insoluble substance by using a slightly alkaline citric acid buffer solution, and adding the slightly alkaline citric acid buffer solution into the collected upper-layer insoluble substance for dissolving;

S7: and (3) dialyzing the solution to a constant volume to obtain a protease extract, and dialyzing the solution added with the alkalescent citric acid buffer solution to a constant volume to obtain the protease extract.

2. The method for producing the protease for promoting the growth of plant roots and fruits according to claim 1, wherein the method comprises the following steps: the residue obtained in S4 is subjected to the following processing steps:

s9: inoculating fluorescent pseudo-single package and lipolytic yeast, and inoculating fluorescent pseudo-single package and lipolytic yeast to the prepared culture medium;

S10: fermenting, namely stirring a culture medium inoculated with fluorescent pseudo-single package and lipolytic yeast and then fermenting;

s11: separating and purifying, namely further separating impurities from the enzyme obtained after the fermentation is finished, and purifying the target enzyme;

s12: mixing to obtain the final product, and mixing the purified target enzyme with the obtained protease extractive solution to obtain the final product.

3. the method for producing the protease for promoting the growth of plant roots and fruits according to claim 1, wherein the method comprises the following steps: the S2: the pretreatment comprises the following steps:

s201: cleaning and removing impurities, cleaning the collected stem leaves, tubers or root tubers to remove the unwanted impurities;

S202: crushing, namely crushing the cleaned and impurity-removed stem leaves, tubers or root tubers by using a crusher, and collecting the crushed stems, tubers or root tubers for later use.

4. The method for producing the protease for promoting the growth of plant roots and fruits according to claim 1, wherein the method comprises the following steps: the S4: the filtration and centrifugation for obtaining the supernatant and the residue comprises the following steps:

s401: filtering the slurry, and filtering the homogenate by a filter screen;

s402: centrifuging the filtrate, and adding the filtered filtrate into a centrifugal device for centrifuging;

s403: collecting supernatant and residues, collecting the supernatant obtained after centrifugation, and collecting the residues obtained by filtration.

5. the method for producing the protease for promoting the growth of plant roots and fruits according to claim 4, wherein the protease is produced by the following steps: the filter screen is a filter screen formed by 8 layers of gauze.

6. the method for producing the protease for promoting the growth of plant roots and fruits according to claim 4, wherein the protease is produced by the following steps: the centrifuge apparatus is a centrifuge rotating at 4000 rpm.

7. a method for producing a plant root and fruit growth promoting protease according to any one of claims 1 to 6, wherein: the S8: the preparation of the culture medium for fermentation comprises the following steps:

s801: preparing materials, namely preparing animal protein, plant protein, inorganic salt and water in a certain proportion;

S802: adding fermentation auxiliary materials into the mixture,

s803: stirring and homogenizing, and stirring the prepared culture medium uniformly by using a stirrer for later use.

8. the method for producing the protease for promoting the growth of plant roots and fruits according to claim 7, wherein the protease is produced by the following steps: and the protease finished product needs to be packaged and stored in a warehouse.

Technical Field

the invention belongs to the technical field of bioengineering, and particularly relates to a method for producing protease for promoting growth of plant roots and fruits.

Background

proteases are enzymes (enzymes) in organisms which are effective in breaking down proteins; the decomposition method is to break peptide bonds that link amino acids into polypeptide chains; small molecule compounds that inhibit protease activity are called protease inhibitors; inhibitors of many viral proteases are very effective antiviral agents; they can be classified into endopeptidases and exopeptidases according to their way of hydrolyzing polypeptides. Endopeptidases cleave the protein molecule internally, forming smaller molecular weight peptides and peptones. Exopeptidases hydrolyze peptide bonds one by one from the free amino or carboxyl terminus of the protein molecule to free amino acids, aminopeptidases and carboxypeptidases. Proteases can be further classified into serine proteases, thiol proteases, metalloproteases and aspartic proteases according to their active center and optimum pH. According to the optimum pH value of the reaction, the protease is divided into acid protease, neutral protease and alkaline protease. Proteases used in industrial production are mainly endopeptidases. Proteases are widely found in animal viscera, plant stems and leaves, fruits and microorganisms. Microbial proteases are produced mainly by molds, bacteria, and secondly by yeasts and actinomycetes. Enzymes that catalyze the hydrolysis of proteins. There are many types, and pepsin, trypsin, cathepsin, papain, subtilisin, and the like are important. Proteases have strict selectivity for the reaction substrate on which they act, and a protease can act only on certain peptide bonds in a protein molecule, such as peptide bonds formed by catalytic hydrolysis of basic amino acids by trypsin. The protease has wide distribution, is mainly present in human and animal digestive tracts, and is rich in plants and microorganisms. Because of the limited resources of animals and plants, the protease preparation is mainly prepared by fermenting the microorganisms such as bacillus subtilis, aspergillus terreus and the like in the industrial production.

The harvest is affected and the yield of the fruits is greatly reduced because of insufficient growth in the growth process of plant roots and fruits, so that the problem that a plurality of growers need to solve is to promote the growth of the plant roots and the fruits.

disclosure of Invention

The invention aims to: in order to solve the problems that the yield of the plant roots is affected and the yield of fruits is greatly reduced because the plant roots are not fully grown in the growth process of the fruits, the production method of the protease for promoting the growth of the plant roots and the fruits is provided.

The technical scheme adopted by the invention is as follows:

A method for producing protease for promoting growth of plant roots and fruits comprises the following steps:

s1: collecting stems and leaves of plants of the same or same genus, and collecting stems, leaves, tubers and root tubers of plants of the same or same genus;

S2: pre-treating, namely cleaning and crushing the collected stem leaves, tubers or root tubers;

S3: adding phosphate buffer solution, stirring and homogenizing, adding phosphate buffer solution into the crushed material, and fully and uniformly stirring to form slurry;

s4: filtering and centrifuging to obtain supernatant and residue, filtering the homogenate, and centrifuging the filtration to obtain supernatant and residue;

S5: salting out supernatant with ammonium sulfate, centrifuging to collect upper insoluble substance, adding ammonium sulfate into supernatant, salting out, centrifuging, and collecting upper insoluble substance;

s6: dissolving the upper-layer insoluble substance by using a slightly alkaline citric acid buffer solution, and adding the slightly alkaline citric acid buffer solution into the collected upper-layer insoluble substance for dissolving;

s7: dialyzing the solution to a constant volume to obtain a protease extract, and dialyzing the solution added with the alkalescent citric acid buffer solution to a constant volume to obtain the protease extract;

s8: preparing a culture medium for fermentation according to a certain proportion as required;

s9: inoculating fluorescent pseudo-single package and lipolytic yeast, and inoculating fluorescent pseudo-single package and lipolytic yeast to the prepared culture medium;

s10: fermenting, namely stirring a culture medium inoculated with fluorescent pseudo-single package and lipolytic yeast and then fermenting;

s11: separating and purifying, namely further separating impurities from the enzyme obtained after the fermentation is finished, and purifying the target enzyme;

s12: mixing to obtain the final product, and mixing the purified target enzyme with the obtained protease extractive solution to obtain the final product.

wherein the S2: the pretreatment comprises the following steps:

s201: cleaning and removing impurities, cleaning the collected stem leaves, tubers or root tubers to remove the unwanted impurities;

S202: crushing, namely crushing the cleaned and impurity-removed stem leaves, tubers or root tubers by using a crusher, and collecting the crushed stems, tubers or root tubers for later use.

wherein the S4: the filtration and centrifugation for obtaining the supernatant and the residue comprises the following steps:

s401: filtering the slurry, and filtering the homogenate by a filter screen;

S402: centrifuging the filtrate, and adding the filtered filtrate into a centrifugal device for centrifuging;

S403: collecting supernatant and residues, collecting the supernatant obtained after centrifugation, and collecting the residues obtained by filtration.

Wherein, the filter screen is the filter screen that 8 layers of gauze are constituteed.

wherein the centrifugal equipment is a centrifugal machine with the rotating speed of 4000rpm, and the filtrate is centrifuged for 10 min.

wherein the S8: the preparation of the culture medium for fermentation comprises the following steps:

S801: preparing materials, namely preparing animal protein, plant protein, inorganic salt and water in a certain proportion;

S802: adding fermentation auxiliary materials into the mixture,

s803: stirring and homogenizing, and stirring the prepared culture medium uniformly by using a stirrer for later use.

wherein, the protease finished product needs to be packaged and stored in a warehouse.

in summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. in the invention, the stem leaves, tubers and root tubers of the same plants or plants in the same family are selected for collection to produce the protease, so that the growth promoting effect of the produced finished products on plant roots and fruits can be greatly improved, the adaptability is better, and the side effect is less.

2. In the invention, the filtered supernatant and the filtered residues are treated in a subsection way, and the treated substances are fused, so that the production efficiency is greatly increased, and the production cost is reduced.

3. in the invention, the slurry which is prepared by adding the phosphate buffer solution into the crushed material and fully and uniformly stirring the mixture into slurry is filtered by 8 layers of gauze filter screens, so that the filter effect is good, the residue is less, and the supernatant in the filtrate can be conveniently extracted subsequently.

4. in the invention, the centrifugal equipment is a centrifugal machine with the rotating speed of 4000rpm, the filtrate is centrifuged for 10min, the separation is thorough, and the supernatant is easy to absorb.

Drawings

FIG. 1 is a block diagram schematically illustrating a first embodiment of the present invention;

FIG. 2 is a block diagram schematically illustrating the structure of a second embodiment of the present invention;

FIG. 3 is a block diagram schematically illustrating the structure of S2 in the present invention;

FIG. 4 is a block diagram schematically illustrating the structure of S4 in the present invention;

fig. 5 is a block diagram schematically illustrating the structure of S8 in the present invention.

Detailed Description

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

in the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention; the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance; furthermore, unless expressly stated or limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, as they may be fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.