CN108753631B - Trichoderma reesei and application thereof in tannase production - Google Patents

Abstract

The invention relates to a mutant strain trichoderma reesei for high yield of tannase, which is preserved in China center for type culture collection of Wuhan university in Wuhan, China in 6 and 21 months in 2018, and the preservation number is CCTCC NO: M2018389. The invention also provides application of the trichoderma reesei mutant strain in producing tannase. The Trichoderma reesei mutant strain can greatly improve the expression amount of tannase, after fermentation for 160h in a 20L tank, the tannase activity in the mutant strain fermentation supernatant reaches 158u/ml, and is improved by 56.4% compared with the original strain, so that unexpected technical effects are achieved. The Trichoderma reesei mutant strain can be widely applied to production of tannase, so that the production cost of the tannase is reduced, and the popularization and application of the mutant strain in the fields of food and feed processing and the like are promoted.

Description

Trichoderma reesei and application thereof in tannase production

Technical Field

The invention relates to the technical field of genetic engineering, in particular to trichoderma reesei and application thereof in tannase production.

Background

Tannase, known collectively as tannin acyl hydrolase (Tannase, EC 3.1.1.20), hydrolyzes ester and dephenolic carboxyl bonds in gallic acid tannins to produce gallic acid and glucose. Tannase is widely distributed in nature, early reports about tannase are 20 th century, and Freudenberg discovers that the tannase is contained in hypha of Aspergillus niger during culture, and later discovers that various microorganisms including fungi, yeasts and bacteria can produce the tannase; tannase is also present in some plant materials, such as tea extracts. The tannase can be widely applied to the fields of food industry, fine chemical industry and the like.

In the production of liquid tea and instant tea, polyphenols, caffeine, proteins and the like form a complex to form what is called "turbid after cooling", and therefore a "transfer dissolution" process is required. Tannase is the specific enzyme for enzymatic resolution. It can break ester bond between catechol and gallic acid, hydrolyze bitter ester catechin, and release gallic acid anion to compete with theaflavin and thearubigin for caffeine to form water soluble short chain substance with small molecular weight, thereby reducing turbidity of tea soup. The tannase is applied to deep processing of black tea, green tea and oolong tea, and can increase the content of soluble metal elements in the tea. With the increasing and deepening application of tannase in the tea beverage industry, the U.S. Food and Drug Administration (FDA) has confirmed tannase as a safe product.

Tannase is also useful in clarification of beer and fruit juice, prevention of deterioration caused by phenols in fruit wine and fruit juice, production of soft drink having coffee flavor, stabilization of malt polyphenol, improvement of wine flavor, and the like. Korea scientists Chae Soo-Kyu and Yu Tai-Jong treated acorn with tannase to make it edible; the rice flour and acorn powder are mixed at a ratio of 1:1, and then the mixture is acted by amylase and tannase to brew acorn wine. Tannase can also be used to remove the bitter and astringent taste of natural foods due to tannin.

In addition, tannase is also widely used in the field of feed industry. Plant-derived feeds often contain tannins, which can coagulate and precipitate plant proteins and digestive enzyme proteins secreted by livestock themselves, thereby reducing the digestibility of ingested proteins by livestock and increasing the nitrogen source in fecal excretion. Therefore, the corresponding treatment of tannase in the feed processing process can greatly improve the utilization rate and the absorption rate of the plant protein of the livestock and reduce the production cost of the animal husbandry. The research of Viveros A and the like finds that the tannase is added into the bean and shell-changing feed for the domestic chicken, so that the chicken acquisition amount is remarkably increased.

Tannase is mainly produced by microorganisms, and strains capable of producing tannase are rich, and besides traditional penicillium, aspergillus and yeast, there are pasteurella, endophytic fungi, trichoderma viride, fusarium solani and the like. At present, the types of commercial tannase products are few, and the enzyme price is high due to low yield of production strains, so that the wide application of the enzyme in the fields of food, feed and the like is severely limited. Therefore, the development of a producing strain with high tannase productivity is imminent.

Disclosure of Invention

The invention provides a Trichoderma reesei (Trichoderma reesei) strain for high yield of tannase, aiming at solving the problems in the prior art. The applicant firstly constructs and obtains a trichoderma reesei engineering strain for recombinant expression of tannase, and then obtains a mutant strain with remarkably improved tannase yield by screening through an ultraviolet mutagenesis method, and the mutant strain can be widely applied to production of the tannase.

The invention provides a Trichoderma reesei engineering strain, which carries a recombinant plasmid for recombinant expression of tannase genes.

The amino acid sequence of the tannase is SEQ ID NO. 1, and the coding nucleotide sequence is SEQ ID NO. 2.

The invention provides a mutant strain Trichoderma reesei 4QT2(Trichoderma reesei 4QT2) which is preserved in China center for type culture Collection of Wuhan university in China at 6 and 21 months in 2018, wherein the preservation number is CCTCC NO: M2018389.

The invention also provides application of the trichoderma reesei mutant strain in producing tannase.

The tannase gene derived from Aspergillus oryzae (Aspergillus oryzae) is expressed in Trichoderma reesei (Trichoderma reesei) host cells to construct a recombinant strain Trichoderma reesei 4QT, and after fermentation is carried out in a 20L tank for 160h, the tannase enzyme activity in fermentation supernatant reaches 101 u/ml. The applicant takes Trichoderma reesei 4QT as an original strain, and obtains a mutant strain Trichoderma reesei 4QT2 through multiple rounds of ultraviolet mutagenesis and final screening, the expression level of tannase can be greatly improved, after fermentation is carried out for 160h in a 20L tank, the tannase activity in the mutant strain fermentation supernatant is up to 158u/ml, and is improved by 56.4% compared with the original strain, and unexpected technical effects are obtained. The Trichoderma reesei strain can be widely applied to production of tannase, thereby being beneficial to reducing the production cost of the tannase and promoting the popularization and application of the Trichoderma reesei strain in the fields of food and feed processing and the like.

Drawings

FIG. 1 is a map of plasmid pTG;

FIG. 2 is a 20L tank fermentation process curve;

FIG. 3 is an SDS-PAGE protein electrophoresis: wherein: m is a protein molecular weight Marker, and lanes 1 and 2 are respectively mutant strain trichoderma reesei 4QT2 and starting strain trichoderma reesei 4QT fermentation supernatant; the 65kDa protein band indicated by the arrow is tannase.

Detailed Description

The present invention uses conventional techniques and methods used IN the fields of genetic engineering and MOLECULAR BIOLOGY, such as the methods described IN MOLECULAR CLONING, A LABORATORY MANUAL,3nd Ed. (Sambrook,2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the field on the basis of the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention.

The present invention will be described in detail with reference to specific embodiments.

EXAMPLE 1 cloning of tannase Gene and construction of recombinant plasmid

The applicants performed codon optimization of tannase gene derived from Aspergillus oryzae (Aspergillus oryzae) according to the codon preference of Trichoderma reesei, adding TCTAGA (Xba I cleavage site) of 6 bases before the 1 st amino acid codon, adding TCTAGA (Xba I cleavage site) after the termination codon TAA thereof, the optimized nucleotide sequence being SEQ ID NO:2, synthesized by Shanghai Jersey company, and the encoded amino acid sequence being SEQ ID NO: 1.

The tannase gene was amplified by PCR. The primer sequences are as follows:

primer 1 (F): GCTCTAGA ATGCGCCAGCACTCCCGCATG

Primer 2 (R): GCTCTAGA TTAGTAGACGGGGACCTTGAA

The PCR reaction conditions are as follows: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30s, renaturation at 56 ℃ for 30s, extension at 72 ℃ for 110s, and after 30 cycles, heat preservation at 72 ℃ for 10 min. Agarose gel electrophoresis results show that the tannase gene size is 1767bp fragment.

The tannase gene fragment and the expression plasmid pTG obtained above are subjected to single restriction enzyme digestion by restriction enzyme XbaI under the following conditions:

carrying out enzyme digestion treatment for 2h in water bath at 37 ℃, respectively recovering two target fragments after electrophoresis, and dissolving in 20ul ddH₂And O. Ligation was performed with T4DNA ligase in the following system:

connecting for 1h at 22 ℃, transforming escherichia coli DH5a competence, coating an LB + AAP plate, culturing overnight at 37 ℃, growing a single colony, verifying the correctly connected transformant by colony PCR, extracting plasmid, sequencing, and obtaining the recombinant plasmid pTG-Tan containing tannase gene after sequencing is correct.

Example 2 construction of recombinant strains of Trichoderma reesei

1. Preparing protoplasts:

inoculating Trichoderma reesei host cells to a PDA + U (potato 200g/L, boiling for 20-30min, filtering to remove residues, glucose 2%, Uridine 1%, agar powder 1.5%) plate, and culturing at 30 deg.C for 5-7 d; cutting 2cm × 2 cm-sized fungus block, inoculating into 100ml liquid PDA + U (potato 200g/L, boiling for 20-30min, filtering to remove residue; glucose 2%; Uridine 1%) culture medium, and culturing at 30 deg.C for 16 hr to grow mycelium for transformation; after the grown mycelia were filtered, it was resuspended in 20ml of 1.2M magnesium sulfate solution; adding 0.2g of lysozyme, culturing at 30 ℃ and 100rpm for 2-3 h; filtering the cracked mycelium with 2 layers of mirror paper, centrifuging at 3000rpm for 10min to obtain protoplast; filtering the cracked mycelium with a piece of lens wiping paper, and centrifuging to obtain a protoplast; then, the mixture is resuspended by using a proper amount of sorbitol solution.

2. And (3) transformation:

washing the obtained Trichoderma reesei protoplast with 1.2M sorbitol solution for 2 times, and re-suspending with appropriate amount of sorbitol solution to make the protoplast concentration reach 10⁸Per ml; adding 10ul of the prepared recombinant vector pTG-Tan into 200ul of protoplast, adding 50ul of 25% PEG6000, ice-cooling for 20min, adding 2ml of 25% PEG6000, and standing at room temperature for 5 min; adding 4ml sorbitol solution, mixing, pouring 50ml conversion upper layer culture medium, pouring into 4 conversion lower layer flat plates, solidifying the upper layer culture medium, and culturing in 30 deg.C incubator for 5 d.

3. And (3) transformant screening:

after 5 days of culture, the grown colonies are picked up, spotted on a transformation lower layer plate for re-screening, and cultured for 3 days at 30 ℃. The transformants which grew normally were inoculated into fresh PDA plates, respectively, and cultured at 30 ℃ for 5-7 days. Each transformant was harvested into 2cm × 2 cm-sized clumps, inoculated into 50ml of liquid shake flask medium (1% glucose, 2% lactose, 1.5% corn steep liquor, 0.9% ammonium sulfate, 0.15% magnesium sulfate, 0.073% citric acid, 0.1125% calcium chloride, 0.1% trace elements) respectively, fermented at 28 ℃ for 5 days. After culturing for 5 days, centrifuging the thalli to obtain supernatant, namely crude enzyme liquid, and respectively carrying out SDS-PAGE protein electrophoresis detection and tannase enzyme activity detection.

Detecting the activity of the tyrosinase in the fermentation supernatant of the positive transformant, and screening out the positive transformant with the highest enzyme activity, which is named as Trichoderma reesei 4QT (Trichoderma reesei 4 QT).

Example 3 mutagenesis screening

The mutation caused by ultraviolet mutagenesis has strong randomness, and the effect generated by mutation is random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, the screening workload is large, and the possibility that effective positive mutations cannot be obtained exists. However, ultraviolet mutagenesis requires simple equipment and low cost, and can obtain a large number of mutants in a short time, so that it is still a common mutagenesis breeding method.

The applicant takes Trichoderma reesei 4QT as an original strain, and carries out genetic modification on the original strain by an ultraviolet mutagenesis method, thereby further improving the yield of tannase.

1. Determination of the lethality rate:

inoculating trichoderma reesei 4QT to a PDA plate, and culturing at 30 ℃ for 5-7 d. When a large amount of spores are generated on the surface of the colony, 5ml of sterile water is absorbed for elution to obtain a spore liquid, the spore liquid is resuspended by the sterile water after centrifugation, and a blood counting chamber is used for counting. A90 mm petri dish was taken and 5ml of diluted spore suspension (concentration 1X 10) was added⁷) Adding a rotor and stirring on a magnetic stirrer to make the spore liquid in a uniform state. Irradiating with ultraviolet lamp with power of 9w at a vertical distance of 20cm in a sterile ultra-clean bench for 30s, 45s, 60s, 75s, 90s, 105s and 120s, diluting the irradiated spore solution for 10, 100 and 1000 times, coating 100ul PDA plate, culturing at 30 deg.C for 2-3d, counting, and calculating lethality with unirradiated spore solution as control. Wherein the lethality is 95% when the irradiation time is 90s, and the irradiation time is selected for subsequent mutagenesis experiments.

2. First round mutagenesis screening:

a90 mm petri dish was taken and 5ml of diluted spore suspension (concentration 1X 10) was added⁷) Adding a rotor and stirring on a magnetic stirrer to make the spore liquid in a uniform state. Irradiating with ultraviolet lamp with power of 9w in sterile ultra-clean bench at vertical distance of 20cm for 90s, diluting 1000 times, coating 100ul PDA plate, and culturing at 30 deg.C for 2-3 d.

Totally coating 200 PDA plates, culturing at 30 ℃ for 2-3d, growing 30-50 colonies on each plate, and screening short-branched mutants through colony morphology. The applicant selects 75 mutant bacteria with small colony morphology, dense hyphae and short villus around the colony, and the mutant bacteria are respectively inoculated to a PDA plate and cultured for 5-7 days at 30 ℃. Each transformant was cut into 2cm × 2cm pieces, inoculated into 50ml liquid shake flask medium, fermented, and cultured at 28 deg.C for 5 days. After culturing for 5 days, centrifuging the thallus to obtain supernatant, namely crude enzyme liquid, and respectively carrying out protein electrophoresis detection and tannase enzyme activity detection.

The result shows that the enzyme activity of tannase in the supernatant enzyme fermented by no mutant strain in 75 mutant strains obtained by the first round of ultraviolet mutagenesis screening is higher than that of the original strain; wherein, the enzyme activity of 46 mutant strains is basically equivalent to that of the original strain, and the enzyme activity of the other 29 mutant strains is reduced by 6-18 percent even compared with that of the original strain.

The applicant carries out 8 rounds of mutagenesis screening according to the method, finally obtains 3 mutant strains with tannase yield remarkably higher than that of the original strain, and the mutant strains are named as Trichoderma reesei 4QT1, Trichoderma reesei 4QT2 and Trichoderma reesei 4QT3 respectively, and the tannase yield of the mutant strains is respectively improved by 35%, 58% and 61% compared with the original strain.

(1) Definition of tannase enzyme Activity Unit

At 30 deg.C and pH 5.0, degrading Propyl Gallate (PG) solution per minute releases the amount of enzyme required to produce 1 μmol gallic acid, defined as one unit of enzyme activity U.

(2) Enzyme activity measuring method

Gallic acid standard solution (10 mmol/L): 0.18813g of gallic acid is weighed out, dissolved in a disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0, and the volume is determined to be 100 ml.

Propyl gallate (10 mmol/L): weighing 0.2122g propyl gallate, adding 80ml disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0, heating until completely dissolving, cooling, adjusting pH to 5.0, and diluting to 100 ml.

Rhodanine solution (0.667%): weighing 0.667g of rhodanine, adding 80ml of methanol for dissolving, and metering to 100ml with methanol after dissolving to prepare the final product.

Drawing a gallic acid standard curve: preparing gallic acid standard solutions with different concentrations by using disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0, and preparing 9 different concentration gradients from 40-240 mu mol/L. Mixing 0.5ml gallic acid standard solution with 0.3ml methanol rhodanine, adding into all test tubes, water bathing at 30 deg.C for 5min, adding 4.2ml KOH solution (0.5mol/L), and keeping at 30 deg.C for 10 min. The absorbance at 520nm was measured using buffer instead of standard solution as a blank (A520). A standard curve is drawn by taking the concentration of gallic acid (mmol/L) as an abscissa and taking A520 as an ordinate.

And (3) determination: before the reaction starts, 10ml of propyl gallate solution and the enzyme solution to be detected are subjected to heat preservation in a water bath at the temperature of 30 ℃ for 5-10 min.

1) Taking 3 test tubes, namely a blank tube and a test tube respectively, adding 0.25ml of propyl gallate solution into each tube, then adding 0.25ml of enzyme solution to be tested into each test tube respectively, and carrying out water bath reaction at 30 ℃ for 5 min.

2) 0.3ml of the solution of rhodanine in methanol was added to all tubes and incubated for 5 min.

3) 4.2ml of KOH solution (0.5mol/L) was added to all the tubes, 0.25ml of the enzyme solution was added to a blank tube, the temperature was maintained at 30 ℃ for 10min, the blank tube was zeroed, and the absorbance A520 of the solution was measured at 520nm for each tube.

The enzyme activity calculation formula is as follows:

in the formula:

x is the enzyme activity of the sample, and the unit is U/ml;

a520-difference in blank absorbance of sample;

c0-intercept of standard curve;

0.5-propyl gallate is added with the total volume of the enzyme solution to be detected, and the volume is 0.5 ml;

n is dilution multiple;

k-the slope of the standard curve;

1/0.25-enzyme activity converted into 1ml of enzyme solution;

5-reaction time, 5 min;

example 4 fermentation Scale-Up

The applicant further ferments the original strain Trichoderma reesei 4QT and the mutant strains Trichoderma reesei 4QT1, 4QT2 and 4QT3 in 20L tanks respectively. And when the fermentation is finished for 160h, respectively measuring the enzyme activity of the tannase in the fermentation supernatant. The result shows that the tannase activity in the supernatant of the original strain Trichoderma reesei 4QT fermentation reaches 101u/ml, while the fermentation enzyme activities of the mutant strains Trichoderma reesei 4QT1, 4QT2 and 4QT3 are 135u/ml, 158u/ml and 143u/ml respectively, which are respectively improved by 33.7%, 56.4% and 41.6% compared with the original strain.

The fermentation process curves of the trichoderma reesei 4QT and the mutant trichoderma reesei 4QT2 are shown in figure 2, and after fermentation for 50 hours, the enzyme activity of the fermentation liquor of the mutant trichoderma reesei 4QT2 is obviously higher than that of the original strain; when the fermentation is finished for 160h, the tannase activity in the supernatant obtained by fermenting the original strain trichoderma reesei 4QT reaches 101u/ml, while the tannase activity in the supernatant obtained by fermenting the mutant strain trichoderma reesei 4QT2 reaches 158u/ml, which is improved by 56.4% compared with the original strain.

Meanwhile, after fermentation is finished, SDS-PAGE electrophoresis detection is carried out on fermentation supernatants of the original strain Trichoderma reesei 4QT and the mutant strain Trichoderma reesei 4QT 2. The result is shown in figure 3, the protein band at 65kDa indicated by the arrow is tannase, the content of the tannase in the supernatant obtained by fermenting the mutant strain trichoderma reesei 4QT2 in the Lane 1 is obviously higher than that in the supernatant obtained by fermenting the starting strain trichoderma reesei 4QT in the Lane 2, and unexpected technical effects are achieved.

The applicant has deposited the above mutant strain Trichoderma reesei 4QT2(Trichoderma reesei 4QT2) in 21.6.2018 in the China center for type culture Collection, Wuhan university, Wuhan, China, with the preservation number of CCTCC NO: M2018389.

Sequence listing

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<120> Trichoderma reesei and application thereof in tannase production

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Claims (2)

1. The Trichoderma reesei mutant strain is characterized in that the preservation number of the Trichoderma reesei mutant strain is CCTCC NO: M2018389.

2. Use of the mutant strain of trichoderma reesei of claim 1 for the production of tannase.

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