AU2021101266A4 - A screening method for efficiently isolating endoxylanase producing microorganism and its application - Google Patents

Abstract

The invention provides A screening method for efficiently isolating endoxylanase producing microorganism and its application, which comprises the following steps: (1) Preparing sample source; (2) Screening strains; (3) Preparation of xylanase fermentation broth. According to the invention, firstly, strains are separated and cultured in a growth medium, and then strains are screened by a double-layer plate method, and Azo- Xylan(Birchwood) is used as the screening substrate. This method can solve the problem of mixed colonies in the process of culturing microorganisms first and then adding Congo red solution for color reaction. It can also solve the problem of false positive in the process of adding Congo red in the preparation of plate and then culturing microorganisms.

Description

A screening method for efficiently isolating endoxylanase producing microorganism

and its application

TECHNICAL FIELD

The invention belongs to the field of biotechnology, and particularly relates to a

screening method for efficiently isolating endoxylanase producing microorganism and its

application.

BACKGROUND

Hemicellulose is a very rich resource in nature, accounting for about 35% of the dry

weight of plants. It is widely found in agricultural and sideline products such as corn cob,

wheat bran, rice bran, straw and bagasse, and is second only to cellulose in nature. Xylan

is an important component of hemicellulose, which is a complex and diverse xylan

molecule composed of p-D- pyranose xylose units connected by P-1,4- glycosidic bonds,

with various substituents such as acetyl, arabinose residues and glucose residues on the

main chain. The complete degradation of xylan requires the participation of many enzymes,

including P-1,4- endoxylanase (EC3.2.1.8), j-xylosidase, (EC3.3.1.37) and ferulic acid

esterase (EC3.1.1.73). Among them, p-1,4-endoxylanase acts on the main chain of xylan

to produce different chain lengths of oligosaccharide. It is the most critical enzyme in the

xylan degrading enzyme system. At the same time, it is also a xylan degrading enzyme that

has been widely studied and applied. Xylooligosaccharides produced by enzymatic method

can selectively proliferate bifidobacteria in animal intestines, and play an important role in

reducing cholesterol, maintaining intestinal health and promoting calcium absorption. In addition, xylanase plays an important role in pulping and papermaking, food, feed additives and biotransformation.

At present, Congo red staining method is widely used for screening xylan-degrading

strains, which mainly adopts two ways. One way is to culture microorganisms first, then

add Congo red solution for color reaction, and then decolorize with sodium chloride to

form xylan-degrading transparent rings of different sizes. The advantage of this way is that

the displayed color reaction is basically the function of xylan-degrading bacteria, but the

disadvantage is that it is cumbersome to operate, and adding Congo red solution will cause

confusion among colonies. Another way is to add Congo red when preparing the plate. This

method has the advantages of simple operation and no colony mixed problem. The

disadvantage is that both agar and potato juice contain starch substances, which can make

amylase-producing microorganisms produce false positive reaction. In addition, some

microorganisms with the ability to degrade pigment will degrade Congo red and form

transparent circles in the long-term culture process, which is difficult to distinguish from

xylan-degrading bacteria. According to the invention, firstly, strains are isolated and

cultured in a growth medium, and then strains are screened by a double-layer plate method,

and Azo-xylan(Birchwood) is added as a screening substrate in an upper culture medium,

so that the above problems can be solved, and endoxylanase producing strains can be

screened quickly, accurately and effectively. In addition, the strain Saccharothrix

variisporeaYJ screened by this method is different from the strain screened by previous

reports, and the xylanase produced by the strain can directly hydrolyze corn cob powder to

produce xylo-oligosaccharide, thus omitting the step of preparing xylan by alkali treatment

of corn cob, thus reducing environmental pollution and production cost.

SUMMARY

In view of the above problems in the prior art, Azo-xylan (Birchwood) is used as a

screening substrate, and the double-layer plate method can solve the above problems. Azo

xylan (Bichwood) is a high molecular compound formed by combining xylan from birch

with dye Remazol brilliant blue R (one Remazol Brilliant Blue combines 20 sugar

residues). Xylanase can degrade Azo-xylan into low molecular weight dye compounds and

oligosaccharides. Low molecular weight dye compounds are soluble in ethanol solution

(transparent in the upper plate culture medium), while high molecular weight dye

compounds are precipitated in ethanol solution (blue in the upper plate culture medium).

According to the invention, Azo-xylan is added to the upper plate as a screening substrate,

and a strain producing xylanase generates a transparent circle in the upper plate; and the

method can rapidly, accurately and effectively screen an endoxylanase producing strain.

The invention is realized by the following technical scheme: the screening method for

efficiently isolating endoxylanase producing microorganism and its application comprise

the following steps:

(1) Preparation of sample source: 20g of pig manure and sugarcane leaves were mixed

and composted at a fermentation temperature of 40°C for 3 days, with an initial C/N ratio

of 25:1, a water content of 60% and an initial pH of 8.0 to obtain fermented products;

(2) Strain screening: add lg of the fermented product in step (1) into 1I0mL of sterile

water, then dilute it to 10-6-108 in gradient, apply 100[ 1on the growth medium, culture at

°C for 3-4 days, pour the upper layer of screening medium, continue to culture at 35°C

for Id, add 2ml of 95% ethanol, select colonies with hydrolysis circle by inoculation needle, and streak culture. It is named SaccharothrixvariisporeaYJ and is now preserved in Guangdong Microbiological Collection Center with the preservation number of

GDMCC NO:60442;

(3) Preparation of xylanase fermentation broth: the pure culture strain screened in step

(2) was inoculated into 20ml growth medium and cultured overnight at 35°C and 160rpm

as seed solution for preparing xylanase fermentation broth; 1ml of seed solution was

inoculated into 100ml of sterile fermentation medium, which was cultured at 40°C, 160rpm

for 6 days, then centrifuged at 12000rpm for 5 minutes, and the supernatant was collected

to obtain the fermentation solution for xylanase production;

(4) The xylanase fermentation broth was used to degrade the natural substrate to 150

mesh corncob, and after 5-7h treatment, reducing sugar could be obtained from corncob.

Further, the preparation method of the growth medium is as follows: NaNO3 2.0g,

K2HPO4 1.0g, KCl 0.5g, MgSO4 0.5g, FeSO4 0.01g, sucrose 10g, agar powder 15g, ddH2

o constant volume to 1000ml, sterilization at 115°C for 25min.

And the preparation method of the upper screening medium is as follows: 1g of agar

powder, 20ml of azo-xylan, and the phosphate buffer (20mM, pH8.0) is fixed to 100ml.

Furthermore, the preparation method of the fermentation culture solution is as

follows: NaNO3 2.0g, K2HPO4 1.0g, KCl 0.5g, MgSO4 0.5g, FeSO4 0.01g, corncob 4.Og

are sievedby 60 meshes, the volume is fixed to 1000ml, and sterilized at 1150 C for 25min.

Identification of SaccharothrixvariisporeaeYJ strain:

Genome was extracted by bacterial extraction kit, and 16S rDNA gene was amplified

by PCR: 50 1 system, 251 PrimerSTAR Max DNA polymerase (2x), 1 1 template, 1 1

each of primers F (5'-AAAttTTTTTTCCCCCCGG-3') and R (3'

GGGCCCCTTTAAAAAAAAAC-5'), 22 1 ddH20 The reaction procedure: 98°C for 4

min, 98°C for 10 sec, 55°C for 10 sec, 72°C for 1 min for 30 cycles, and 72°C for 10min.

The PCR products were sent to Huada Gene for sequencing.

After DNA extraction, PCR amplification, sequence sequencing and comparison, it

was found that the 16S rDNA sequence of this strain had the highest homology with

Saccharothrixvariisporea,which was 99.33%. The strain was preliminarily identified as

Saccharothrix variispora, and named as Saccharothrix variisporea YJ (preserved in

Guangdong Microbial Culture Collection Center, with the preservation number of 60442).

And the sequence is as follows:

GGGCTTCGGGTGTTACCGACTTTCGTGACG TGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAG CGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTC GAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTGTGGGAT TCGCTCCACCTCACGGCTTAGCAGCCCTCTGTACCGGCCATTGT AGCATGTGTGAAGCCCTGGACATAAGGGGCATGATGACTTGAC GTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCCATGAGTCCCCGC CATAACGCGCTGGCAACATGGAACGAGGGTTGCG CTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC GACAGCCATGCACCACCTGTACACCAGTCCGAAGAGGCCTACA TCTCTGCAGGTTTCCGGTGCATGTCAAGCCCAGGTAAGGTTCTT CGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGG CCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCC CAGGCGGGGTGCTTAATGCGTTAGCTGCGGCACGGAGAACGTG GAAGTCCCCCACACCTAGCACCCACCGTTTACGGCGTGGACTA CCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCA GCGTCAGTATCGGCCCAGAGACCCGCCTTCGCCACCGGTGTTC CTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGT CTCCCCTGCCGAACTCAAGTCTGCCCGTATCGACCGCAGGCTCCACGTTAAGC GTGAAGTTTTCACGGCCGACGCAACAAACCGCCT ACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTCGCACCCT ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTT CTGCAGGTACCGTCACTCACGCTTCGTCCCTGCTGAAAGAGGT TTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATC AGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTA GGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCT CTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATCACCCCA CCAACAAGCTGATAGGCCGCGGGTCCATCCCGTACCGCCGGAA CTTTCCACCACCACGGATGCCTGAAGTGGTCATATCCGGTATTA GACCTAGTTTCCCAGGCTTATCCCAGAGTACAGGGCAGGTTACC CACGTGTTACTCACCCGTTCGCCGCTCGTGTACCCCGAAGGGCC.

Beneficial effects:

(1) In the present invention, firstly, strains are isolated and cultured in a growth

medium, and then strains are screened by using a double-layer plate method, and Azo- xylan (Birchwood) is added as a screening substrate in the upper culture medium. This method can solve the problem that colonies are mixed in the process of color reaction after culturing microorganisms, and can also solve the problem of false positive in the process of adding Congo red when preparing a plate.

(2) The strain SaccharothrixvariisporeaYJ screened by this method is different from

the strains screened by previous reports. The xylanase produced by this strain can directly

hydrolyze corn cob powder to produce xylo-oligosaccharide, and the step of preparing

xylan by alkali treatment of corn cob is omitted, thus reducing environmental pollution and

production cost.

(3) The fermentation broth of SaccharothrixvariisporeaYJ can degrade the natural

substrate 150 meshes of corncob. After 6h treatment, reducing sugar (0.88mg) can be

obtained from corncob, which is 22 times (0.04mg) of xylooligosaccharide obtained after

extracting xylan from corncob by alkali treatment and then treating the fermentation broth

for 6h.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is the screening of strains producing endoxylanase;

Fig. 2 shows the effect of temperature on xylanase activity and stability;

Fig. 3 shows the effect of pH on enzyme activity.

DESCRIPTION OF THE INVENTION

Example 1

Screening of xylanase-producing strains

Samples from mixed compost of pig manure and sugarcane leaves were diluted,

coated in growth medium, cultured at 35°C for 3 days, and then poured into the upper

screening medium. As shown in the attached Figure 1A of the specification, a transparent

circle can be formed around the strain producing endoxylanase. Colonies were picked with

sterile inoculation needles, streaked, cultured at 35°C for 3 days, and then re-screened. The

results showed that there were obvious hydrolysis circles around the colonies as shown in

Figure 1B of the specification.

Preparation of xylanase fermentation broth

(1) Select the screened pure culture strain, inoculate it into 20ml sterile growth

medium, and cultivate it overnight at 35°C and 160rpm as the seed solution for preparing

xylanase fermentation liquor;

(2) 1ml of seed solution was inoculated into 100ml of sterile fermentation medium,

which was cultured at 40°C and 160 rpm for 6 days, then centrifuged at 12000rpm for 5

minutes, and the supernatant was the fermentation broth for xylanase production.

Enzymatic properties of xylanase from fermentation broth

Add 100[ 1of 1% azo-xylan (birch wood) to 100 1 of fermentation broth, water bath

at 55°C for 2min, centrifuge at 8000rpm for 5min, add 500 1 of 95% ethanol, mix well,

then stand at room temperature for 5min, and measure the light absorption value of

supernatant at 590nm. The crude enzyme solution was treated at 100°C for 10min as

negative control, and three samples of enzyme solution were parallel.

(1) Optimum reaction temperature: measure its enzyme activity according to the

above method at the reaction temperature of 30-65°C, and obtain its optimum reaction

temperature (taking the highest enzyme activity as 100%).

(2) Optimal reaction pH: Adjust the pH of fermentation broth to pH 6.0-8.0(100mM

phosphate buffer), pH 7.5-9.0(100mM Tris-HClbuffer) andpH 8.5-10.0(100 mM glycine

NaOH buffer), and then measure the xylanase activity of crude enzyme solution at the

optimal reaction temperature (when the enzyme activity is the highest,

(3) Temperature stability: Keep the fermentation broth at 30, 35, 40, 45, 50, 55, 60,

and 75°C for 3 hours, and then measure the residual enzyme activity under the optimum

reaction temperature and pH conditions to obtain the thermal stability of the enzyme. The

enzyme activity of the enzyme broth stored at 4°C is defined as 100%, as shown in Figure

2 of the specification.

(4) pH stability: put the fermentation broth in buffer solutions with pH4.0, 5.0, 6.0,

7.0, 8.0, 9.0 and 10.0 respectively for 30min, and then measure the residual enzyme activity

under the optimum reaction temperature and pH conditions to obtain the pH stability of the

enzyme. The enzyme activity of the enzyme broth stored at 4°C is defined as 100%, see

Figure 3 of the specification for details.

(5) Degradation of corn cob by strain fermentation broth: weigh 0.05g corn cob

powder with 150 mesh sieve, add 1.0ml fermentation broth, take 200[ reaction solution at

°C for 6h, add 200[ 1DNS, water bath at 100°C for 10min, and measure OD value at

520nm with spectrophotometer.

Xylanase: The fermentation broth can degrade the natural substrate 150 mesh corn

cob, and after 6 hours treatment, 0.88mg of reducing sugar can be obtained from the corn

cob.

Example 2

0.05g of corn cob powder sieved with 150 meshes was weighed, and the corn cob was

treated by conventional alkali extraction. The rest was the same as in Example 1, and the

reducing sugar was 0.04mg.

To sum up, the present invention adopts a double-layer plate method (adding Azo

xylan(Birchwood) as a screening substrate in the upper culture medium) to screen xylanase

producing strains, which can not only solve the problem of intermixing among colonies in

the process of culturing microorganisms first and then adding Congo red solution for color

reaction, but also solve the problem of false positive in the process of adding Congo red

when preparing plates and then culturing microorganisms; In addition, the strain

SaccharothrixvariisporeaYJ screened by this method is different from the strain screened

by previous reports, and the xylanase produced by the strain can directly hydrolyze corn

cob powder to produce xylo-oligosaccharide, thus eliminating the step of preparing xylan

by alkali treatment of corn cob, thus reducing environmental pollution and production cost;

Furthermore, the fermentation broth of the strain can degrade the natural substrate 150

meshes of corncobs, and after 6 hours of treatment, 0.88mg of reducing sugar can be

obtained from corncobs, which is 22 times (0.04mg) of xylooligosaccharide obtained after

extracting comcobxylan by alkali treatment and then treating the fermentation broth for 6

hours.

Claims (4)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A screening method for efficiently isolating endoxylanase producing

microorganism and its application, which is characterized by comprises the following

steps:

(2) Preparation of sample source: 20g of pig manure and sugarcane leaves were mixed

and composted at a temperature of 40°C for 3 days, with an initial C/N ratio of 25:1, a

water content of 60% and an initial pH of 8.0 to obtain products;

(2) Strain screening: add lg of the product in step (1) into 10mL of sterile water, then

dilute it to 10 -6 -10 -8 in gradient, apply 100[ 1on the growth medium, store at 35°C for 3

4days, pour the upper layer of screening medium, continue to store at 35°C for I d, add 2ml

of 95% ethanol, select colonies with hydrolysis circle byneedle, and streak culture. It is

named SaccharothrixvariisporeaYJ and is now preserved in Guangdong Microbiological

Collection Center with the preservation number of GDMCC NO:60442;

(3) Preparation of xylanase broth: the pure productscreened in step (2) was stirred into

ml growth medium and stored overnight at 35°C and 160rpm as seed solution for

preparing xylanase broth; 1ml of seed solution was stirred into 100ml of sterile medium,

which was stored at 40°C, 160rpm for 6 days, then centrifuged at 12000rpm for 5 minutes,

and the supernatant was collected to obtain the solution for xylanase production;

(4) The xylanase broth was used to degrade the natural substrate to 150 mesh corncob,

and after 5-7h treatment, reducing sugar could be obtained from corncob.

2. The screening method for efficiently isolating endoxylanase producing

microorganism and its application according to claim 1, the preparation method of the medium is as follows: NaNO3 2.0g, K2HPO4 1.0g, KCl 0.5g, MgSO4 0.5g, FeSO4 0.01g, sucrosel0g, agar powder 15g, ddH2 0 constant volume to 1000ml, sterilization at 115°C for 25min.

3. The screening method for efficiently isolating endoxylanase producing

microorganism and its application according to claim 1, wherein the preparation method of

the upper screening medium is as follows: 1g of agar powder, 20ml of azo-xylan, and the

phosphate buffer (20mM, pH8.0) is fixed to 100ml.

4. According to the screening method and application of an efficient endoxylanase

producing strain of claim 1, the preparation method of the solution is as follows: NaNO3

2.0g, K2HPO4 1.0g, KCl 0.5g, MgSO4 0.5g, FeSO4 0.01g, corncob 4.g are sieved by 60

meshes, the volume is fixed to 1000ml, and sterilized at 1150 C for 25min.

FIGURES

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Figure 1

Figure 2

Figure 3