CN116179368A - Aspergillus oryzae for producing acidic lactase and application thereof - Google Patents

Abstract

The invention provides an aspergillus oryzae (Aspergillus oryzae) strain for producing lactase, which is preserved in the Guangdong province microorganism strain collection (GDMCC) at the year of 2022 and 11 and 28, and the preservation address is Guangzhou and the preservation number is GDMCC No.63004. The Aspergillus oryzae provided by the invention is easy to culture, the lactase has good temperature stability and pH stability under acidic conditions, and the activity is highest when the pH is 4.5 at 60 ℃.

Description

A strain of Aspergillus oryzae producing acid lactase and its application

technical field

The invention relates to the technical field of microorganisms, in particular to a strain of aspergillus oryzae capable of producing lactase and an application thereof.

Background technique

Lactase, namely β-galactoside galactohydrolase (β-galactoside galactohydrolase, E.C.3.2.1.23), has the ability to catalyze the hydrolysis of lactose to generate galactose and glucose and has certain transglycosylation activity. Lactase is widely used in food, medicine, environment and other fields, and it is mainly used to solve lactose intolerance in food and medicine. The natural source of lactase is very rich and widely distributed in animals, plants and microorganisms. However, current research has found that only lactase produced by microorganisms has industrial application value. Among them, molds such as Aspergillus niger and Aspergillus oryzae have not been poisoned in safety tests and are considered safe strains. Lactase produced by mold is an extracellular enzyme, which is easy to separate and extract. Its optimum temperature is generally above 50°C and has good thermal stability.

Aspergillus oryzae is an aerobic fungus belonging to the subphylum Discomycotina and the genus Aspergillus, which was originally isolated from koji used to make sake in Japan. Over the years, it has been used in many countries for the fermentation of different foods such as soy sauce. Although Aspergillus oryzae and Aspergillus flavus belong to the yellow-green group of Aspergillus genus, Aspergillus oryzae does not produce aflatoxin, so it was awarded GRAS certification. Molecular and genetic identification techniques are often unable to distinguish the two fungi, A. flavus and A. oryzae, and ultimately rely on morphological and microscopic characteristics to identify A. oryzae. Affected by different media, Aspergillus oryzae can produce a variety of secondary metabolites, such as terpenes, coumarins, oxidized lipids and fatty acids, etc., which have different biological activities, such as anticancer, cytotoxicity, antibacterial, antibacterial Hypertension and antiviral activity, etc. Aspergillus oryzae is also used as a production strain for many industrial enzymes such as neutral and alkaline proteases, α-amylase, glucoamylase, prolyl endopeptidase, cellulase, asparaginase, lipase, pectin enzymes, β-galactosidase, etc.

Contents of the invention

The first object of the present invention is to provide a strain of Aspergillus oryzae that can produce lactase. The Aspergillus oryzae strain (Aspergillus oryzae) was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on November 28, 2022. The address is 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is GDMCCNo.63004.

The medium used is potato dextrose agar medium (PDA medium), cultivated under aerobic conditions at 25-30°C.

The DNA sequence of the Aspergillus oryzae is SEQ ID NO.1.

The aspergillus oryzae is isolated from tidal flat seawater in Ningbo City, Zhejiang Province.

The second object of the present invention is to provide an application of an Aspergillus oryzae strain in expressing lactase.

The optimal reaction temperature of the lactase expressed by the Aspergillus oryzae is 60°C, and the activity is higher at 40-65°C.

The lactase expressed by the Aspergillus oryzae has good thermal stability at 4-60°C.

The optimum reaction pH of the lactase expressed by the Aspergillus oryzae is 4.5, and the activity is higher when the pH is 4-5.

The lactase expressed by Aspergillus oryzae has good stability when the pH is acidic.

Compared with prior art, the beneficial effect of the present invention is:

The present invention isolates and identifies a lactase-producing Aspergillus oryzae strain, analyzes the genome sequence and constructs a phylogenetic tree, and combines the morphological characteristics and microscopic examination results on the Saccharomyces tsarli agar medium (CYA) to determine The strain is Aspergillus oryzae.

The aspergillus oryzae provided by the invention is easy to cultivate, the lactase has good temperature and pH stability, and has the highest activity when the temperature is 60 DEG C and the pH is 4.5.

Description of drawings

Fig. 1 is the bacterium colony form (front) when aspergillus oryzae bacterial strain is cultivated 5d on PDA solid medium in embodiment 3;

Fig. 2 is the bacterium colony form (reverse side) when aspergillus oryzae bacterial strain is cultivated 5d on PDA solid medium in embodiment 3;

Fig. 3 is the morphological figure of Aspergillus oryzae bacterial strain under the microscope in embodiment 3;

Fig. 4 is the bacterium colony morphology (front side) when aspergillus oryzae bacterial strain is cultivated on CYA solid medium for 14d in embodiment 3;

Fig. 5 is the bacterium colony morphology (reverse side) when aspergillus oryzae bacterial strain is cultivated on CYA solid medium for 14d in embodiment 3;

Fig. 6 is the phylogenetic tree constructed based on the sequence result of Aspergillus oryzae strain in embodiment 4;

Fig. 7 is the impact diagram (optimum reaction temperature of lactase) of different temperatures on lactase enzyme activity in embodiment 5;

Fig. 8 is the impact figure (thermal stability of lactase) of different temperature on lactase enzyme activity in embodiment 5;

Fig. 9 is a figure showing the influence of different pH values on lactase activity in Example 6 (the optimal reaction pH of lactase);

Fig. 10 is a graph showing the effect of different pH values on the enzyme activity of lactase in Example 6 (pH stability of lactase).

Detailed ways

The specific implementation of the present invention will be described in detail below in conjunction with the examples. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

Embodiment 1: Screening and obtaining of Aspergillus oryzae strains.

1. Preparation of culture medium

PDA medium: Wash and peel potatoes, weigh 200g and cut into small pieces, boil in pure water for 30 minutes, filter with eight layers of gauze to remove residues, add water to 1L after cooling down, and keep the pH natural. If it is a solid medium, add 20 g of agar, aliquot and sterilize at 121°C for 20 minutes. 50% Glucose: Weigh 50g of glucose, add water to dissolve and make the volume to 100mL, and sterilize at 115°C for 30 minutes.

PDA solid plate: Cool the sterilized PDA medium to about 60°C, add 20g/L glucose and 20mg/mL X-gal, invert the plate, and set aside after solidification.

CYA solid plate: NaNO ₃ 3.0g/L, K ₂ HPO ₄ 1.0g/L, KCl 0.5g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.01g/L, yeast extract 5.0g/L, sucrose 30.0g/L, agar 15.0g/L. Pour the sterilized CYA medium into the plate, solidify and set aside.

2. Screening and acquisition of strains

Sample collection: Seawater samples were collected on July 13, 2022 from coastal beaches of Ningbo City, Zhejiang Province (E 122°49′51″, N 29°47′52″), and stored in sterile bottles.

Screening and acquisition of bacterial strains: After the samples were serially diluted with sterile water, they were distributed on the PDA solid medium by plate coating, numbered and sealed, and cultured upside down in a biochemical incubator at 30°C for 3-5 days. Through the initial screening of the plate, a blue fungus was found on the plate, which was spotted on the PDA solid medium for enrichment and purification.

Example 2: Determination of lactase activity of Aspergillus oryzae strains.

1. Buffer and reagent preparation

Acetate buffer: absorb 25mL of 2N acetic acid, add about 300mL of water, and adjust the pH to 4.5 with 2N NaOH solution. Transfer the solution to a 500mL volumetric flask and make to volume. 10% Na ₂ CO ₃ solution: Weigh 50g of anhydrous sodium carbonate, dissolve with a small amount of deionized water, and dilute to volume in a 500mL volumetric flask. Substrate: Weigh 185.0mg ONPG (o-nitrophenyl-β-D-galactopyranoside), dissolve it with 40mL acetate buffer, and dilute to volume with a 50mL volumetric flask. It is prepared and used immediately, and kept in a cool and dark place until use , and detect within 2 hours. 2mM o-nitrophenol: Weigh 139.0mg o-nitrophenol into a 500mL volumetric flask, dissolve with 10mL 95% ethanol, dilute to the mark with 1% _Na2CO3 solution, and mix _well .

2. Preparation of o-nitrophenol standard curve

Dilute 2mM o- _nitrophenol with 1% _Na2CO3 solution to 0.10, 0.14 and 0.18mM respectively, in a 1cm cuvette at 420nm, use a suitable spectrophotometer to measure three concentrations of o-nitrophenol standard solution Absorbance, and zero calibration operation with water. The linear regression analysis of the absorbance value of each o-nitrophenol was carried out by three standard solution concentrations of o-nitrophenol (0.10, 0.14 and 0.18mM).

3. Acquisition of Lactase Crude Enzyme Solution

Inoculate the Aspergillus oryzae on the PDA solid plate into the PDA liquid medium, culture at 30°C with shaking at 150r/min for 6 days, centrifuge at 4000r/min and collect the supernatant, which is the crude enzyme solution.

4. Lactase Activity Assay

Pipette 2mL of substrate solution into a series of large test tubes and equilibrate in a 37°C water bath. At zero time, quickly draw 0.5mL of appropriately diluted enzyme solution and add it to the temperature-balanced substrate, mix, and immediately put the test tube back into the water bath for reaction. After 15 minutes of reaction, 2.5 mL of 10% sodium carbonate solution was added to all tubes, mixed quickly, and removed from the water bath. Meanwhile, prepare a blank tube and add 2 mL of substrate, 0.5 mL of deionized water and 2.5 mL of 10% sodium carbonate solution. Add 20 mL of water to the sample and blank tubes and mix thoroughly. Using a suitable spectrophotometer, measure the absorbance of each tube of the sample and the blank in a 1cm cuvette at 420nm, and perform zero calibration with water. Record data and calculate. One lactase unit (ALU) is defined as the amount of enzyme required to produce 1 μmol ONP per minute under the test conditions.

Example 3: Morphological characteristics of Aspergillus oryzae strains.

1. Characteristics of Aspergillus oryzae on PDA solid plate

The isolated and purified Aspergillus oryzae strain was transferred to a PDA solid plate, and after culturing at 30°C for 5 days, the colony morphology was observed and the colony shape and color were recorded; the conidiophores and conidia morphology were observed with a microscope. After 5 days of cultivation, the middle spores of the Aspergillus oryzae colony were grass green to dark green, with white hyphae around them, and the back of the colony was yellowish white to light yellow. The colony morphology is shown in Figure 1. Observed under a microscope, the conidia of Aspergillus oryzae are spherical, the conidiophores are long, and the apical capsule is nearly spherical. The microscopic morphology is shown in Figure 2.

2. Characteristics of Aspergillus oryzae on CYA solid plate

Transfer the isolated and purified Aspergillus oryzae strains to the CYA solid plate, observe the colony morphology after culturing at 25°C for 7-15 days, and record the colony shape and color. When cultured for 3 days, the colonies of Aspergillus oryzae were white, and then began to produce yellow spores, which turned into persistent light brown after 7 days. The back of the colony is light yellow, and the colony morphology is shown in Figure 3.

Example 4: Molecular biological identification of Aspergillus oryzae strains.

1. Extraction of fungal DNA genome

Take the culture solution in Example 2, and after centrifugation, take part of the bacteria and put them in a mortar, add liquid nitrogen to grind them thoroughly, collect the ground bacteria in a centrifuge tube, and put them in a -20°C refrigerator for later use.

Take 200 mg of liquid nitrogen ground bacteria, add 3% CTAB, bathe in water at 65° C. for 45 minutes, and centrifuge at 13000 rpm for ten minutes. Take the supernatant and add an equal volume of phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1), and centrifuge at 13000rpm for ten minutes. Add an equal volume of chloroform:isoamyl alcohol (24:1 volume ratio) to the supernatant, and centrifuge at 13,000 rpm for ten minutes. Add twice the volume of pre-cooled absolute ethanol to the supernatant, let stand at -20°C for 20-30 minutes, and centrifuge at 13000rpm for ten minutes. Pour off the supernatant, add 200 μL of 70% ethanol to wash the precipitate, and centrifuge at 13000 rpm for ten minutes. Remove the supernatant and dry the pellet at room temperature. Add 30 μL of water to completely dissolve the DNA, and store in a -20°C refrigerator.

2. PCR amplification

The extracted strain DNA was used for PCR amplification using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system is: 25 μL of 2×TAQ enzyme, 2 μL of ITS1 primer, 2 μL of ITS4 primer, 5 μL of template, and 16 μL of sterile water. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 60 s; extension at 72°C for 7 min, and storage at 4°C.

3. rDNA-ITS sequencing and analysis

Use the DNA gel rapid purification kit (Beijing Quanshijin Biotechnology Co., Ltd.) to recover and purify the target PCR product, and send the purified PCR product to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequencing. The gene sequence is shown as SEQ ID NO: 1 shown.

The measured sequences were compared in NCBI by BLAST, the sequences were analyzed using Mega 4.0 software, and the phylogenetic tree was constructed by the Neighbor-Joining method, as shown in Figure 4.

Through the phylogenetic tree, it was found that the strain had a very high affinity with Aspergillus flavus EF408 and Aspergillus oryzae SW-DG-b. Combining its morphological characteristics and microscopic examination results on the CYA solid plate, the strain was identified as Aspergillus Aspergillus oryzae of the genus fungus.

The strain XNY-41385 was sent to the Guangdong Microbial Culture Collection Center for preservation. The preservation date is November 28, 2022. The preservation unit address is 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is GDMCCNo. 63004.

Embodiment 5: the influence of temperature on lactase enzyme activity

1. Optimum reaction temperature of lactase

The crude enzyme solution was appropriately diluted, and enzymolysis was carried out at reaction temperatures of 20, 30, 40, 50, 55, 60, 65, 70, and 80°C respectively, and the lactase activity was determined according to the method in Example 2. Taking the enzyme activity measured at the optimum reaction temperature as 100%, the relative enzyme activities at different temperatures were calculated respectively. The relative enzyme activity is shown in the table below:

temperature/℃ 20 30 40 50 55 60 65 70 80 Relative enzyme activity/% 30.9 44.3 74.5 89.9 91.4 100 94.2 48.7 0

The relative enzyme activity change curve is shown in Figure 5. The lactase expressed by Aspergillus oryzae has the highest activity when the reaction temperature is 60°C, and the activity is above 70% in the range of 40-65°C, and the enzyme activity decreases rapidly when the temperature continues to rise.

2. Thermostability of lactase

Take the crude enzyme solution and dilute it appropriately, and heat it for 30 minutes at 4, 20, 30, 40, 50, 60, 65, 70, and 80°C respectively, and then immediately place it in an ice bath to cool it. Lactase activity was measured under the conditions. The relative enzyme activity at different temperatures was calculated by taking the enzyme activity measured at the highest activity at different temperatures as 100%. The relative enzyme activity is shown in the table below:

temperature/℃ 4 20 30 40 50 60 65 70 80 Relative enzyme activity/% 100 99.2 98.5 99.6 97.7 94.5 42.1 0 0

The relative enzyme activity change curve is shown in Figure 5. The lactase expressed by Aspergillus oryzae has good thermal stability in the range of 4-60°C, and the relative enzyme activities are all higher than 90%.

Embodiment 6: the impact of pH on lactase enzyme activity

1. The optimal reaction pH of lactase

Take the crude enzyme solution and dilute it properly, carry out enzymolysis under the environment of pH 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0 respectively at the optimum reaction temperature, and measure the lactase enzyme according to the method in Example 2 live. Taking the enzyme activity measured at the optimum reaction pH as 100%, the relative enzyme activities at different pHs were calculated respectively. The relative enzyme activity is shown in the table below:

The relative enzyme activity change curve is shown in Figure 6. The lactase expressed by Aspergillus oryzae has the highest activity when the reaction pH is 4.5, the activity is above 90% when the pH is 4-5, and can still maintain a certain activity when the pH is low.

2. pH Stability of Lactase

The crude enzyme solution was diluted with buffer solution with pH of 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 respectively, treated in this environment for 30 minutes, and then the enzyme activity of lactase was measured under the optimal reaction conditions. The enzyme activity measured at the highest activity at different pH was taken as 100%, and the relative enzyme activities at different pH were calculated respectively. The relative enzyme activity is shown in the table below:

pH 2.0 2.5 3.0 4.0 5.0 6.0 7.0 8.0 Relative enzyme activity/% 35.3 59.8 82.1 100 95.4 94.6 92.4 90.3

The relative enzyme activity change curve is shown in Figure 6. The lactase expressed by Aspergillus oryzae has the best stability when the reaction pH is 4, the activity is above 90% when the pH is 4-8, and the activity is still above 80% when the pH is 3.

Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the protection scope of the present invention.

sequence listing

Ningbo Xinuoya Marine Biotechnology Co., Ltd.

A strain of Aspergillus oryzae producing acid lactase and its application

2022.11.23

2022-11-23

1

SIP Sequence Listing 1.0

1

555

dna

Artificial Sequence

1

aaggatcatt accgagtgta gggttcctag cgagcccaac ctccccaccc gtgtttactg 60

taccttagtt gcttcggcgg gcccgccatt catggccgcc gggggctctc agccccgggc 120

ccgcgcccgc cggagacacc acgaactctg tctgatctag tgaagtctga gttgattgta 180

tcgcaatcag ttaaaacttt caacaatgga tctcttggtt ccggcatcga tgaagaacgc 240

agcgaaatgc gataactagt gtgaattgca gaattccgtg aatcatcgag tctttgaacg 300

cacattgcgc cccctggtat tccggggggc atgcctgtcc gagcgtcatt gctgcccatc 360

aagcacggct tgtgtgttgg gtcgtcgtcc cctctccggg ggggacggggc cccaaaggca 420

gcggcggcac cgcgtccgat cctcgagcgt atggggcttt gtcacccgct ctgtaggccc 480

ggccggcgct tgccgaacgc aaatcaatct tttccaggtt gacctcggat caggtaggga 540

tacccgctga actta 555.

Claims (9)

1. An aspergillus oryzae (Aspergillus oryzae) strain producing lactase, which was deposited at the cantonese province microbiological bacterial collection center (GDMCC) at day 28 of year 2022 under the deposit number GDMCC No.63004.

2. The lactase-producing aspergillus oryzae of claim 1, wherein the culture medium is Potato Dextrose Agar (PDA) medium and is aerobically cultured at 25-30 ℃.

3. The lactase-producing aspergillus oryzae of claim 1 wherein the DNA sequence is set forth in SEQ ID No.1.

4. The lactase-producing aspergillus oryzae of claim 1, wherein the aspergillus oryzae is isolated from beach seawater in Ningbo city of Zhejiang province.

5. Use of a strain of aspergillus oryzae according to any one of claims 1-4 for expression of lactase.

6. The use of a strain of aspergillus oryzae according to claim 5, wherein the lactase expressed by the aspergillus oryzae has an optimal reaction temperature of 60 ℃ and a high activity at 40-65 ℃.

7. The use of a strain of Aspergillus oryzae according to claim 5, wherein the lactase expressed by the Aspergillus oryzae has good thermostability at 4-60 ℃.

8. The use of a strain of Aspergillus oryzae according to claim 5, wherein the lactase expressed by the Aspergillus oryzae has a pH of 4.5 and a higher activity at a pH of 4-5.

9. The use of aspergillus oryzae according to claim 5, wherein the lactase expressed by the aspergillus oryzae has good stability at a pH of 4-8 and the treated activity is still above 80% at a pH of 3.

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