CN107760606B - Trichoderma reesei mutant strain and application thereof - Google Patents

Trichoderma reesei mutant strain and application thereof Download PDF

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CN107760606B
CN107760606B CN201610688711.1A CN201610688711A CN107760606B CN 107760606 B CN107760606 B CN 107760606B CN 201610688711 A CN201610688711 A CN 201610688711A CN 107760606 B CN107760606 B CN 107760606B
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fermentation
mass
trichoderma reesei
activity
strain
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CN107760606A (en
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吴佳鹏
周利伟
王华明
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Weifang KDN Biotechnology Co., Ltd.
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Weifang Kdn Biotech Co ltd
Qingdao Vland Biotech Group Co Ltd
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Abstract

The invention relates to the field of microorganisms, and particularly relates to a trichoderma reesei mutant strain and application thereof. The results of shake flask fermentation and fermentation tank fermentation show that compared with the original strain, the trichoderma reesei mutant strain obviously improves the yield of the non-starch polysaccharase, and can be widely applied to the production of the non-starch polysaccharase, thereby being beneficial to reducing the production cost of the non-starch polysaccharase and promoting the popularization and application of the mutant strain in the field of feed.

Description

Trichoderma reesei mutant strain and application thereof
Technical Field
The invention relates to the field of microorganisms, and particularly relates to a trichoderma reesei mutant strain and application thereof.
Background
Non-starchy polysaccharides (NSP) are plant tissues made of various monosaccharides and uronic acids linked together via glycosidic bonds, mostly branched chain structures, often combined with inorganic ions and proteins, the major components of cell walls, generally difficult to hydrolyze by digestive enzymes secreted by monogastric animals.
The non-starch polysaccharide enzyme mainly comprises various glycosidases, improves the utilization rate of animal to feed nutrients by eliminating the anti-nutritional effect of the non-starch polysaccharide in the feed, and can obtain the best use effect when the proper proportion of the enzyme activity is consistent with the composition of the non-starch polysaccharide in the feed.
The cellulase can break the cell wall rich in fiber, release and utilize the nutrient substances such as protein, starch and the like contained in the cell wall, and degrade the fiber into reducing sugar which can be digested and absorbed by livestock and poultry bodies, thereby improving the utilization rate of the feed. The microorganisms that produce cellulases are much studied by fungi, and few studies are made on bacteria and actinomycetes. Microorganisms currently used for producing cellulase are mainly Trichoderma, Aspergillus niger, Penicillium and Rhizopus, and in addition, Myrothecium, Ruminobacter abyssinicus, Cellophilus, Cellulus flavus, Clamyxobacter laterosporus, Clostridium clostridii, etc. can also produce cellulase.
Xylanase is a specific degrading enzyme of xylan, belongs to a hydrolase and comprises three types, namely endo-xylanase, exo-xylanase and xylosidase. The xylanase producing capability of trichoderma, aspergillus and bacteria is researched more at home and abroad, and the xylanase producing strains which are commercialized at present mainly belong to the trichoderma and aspergillus.
β -glucanase can degrade β -1, 3 and β -1,4 glucoside chains in β -glucan molecules, so that the glucan molecules are degraded into small molecules, lose hydrophilicity and viscosity, change the characteristics of intestinal contents of monogastric animals, the activity of digestive enzymes, the action environment of intestinal microorganisms and the like, microorganisms secreting β -glucanase are bacteria, fungi are mainly fungi, and the fungi mainly comprise trichoderma koningii, trichoderma reesei, trichoderma viride, aspergillus oryzae, mucor mucedinatum, aspergillus niger and the like.
Pectinase is a generic term for enzymes that break down pectin, and is also a multienzyme complex, which generally includes three enzymes, protopectinase, pectate pectin hydrolase, and pectinase. The combined action of these three enzymes allows complete breakdown of the pectin. The strains for industrial production of pectinase are mainly moulds, and the commonly used strains comprise aspergillus wenshuni, penicillium malaccensis, aspergillus niger, rhizoctonia solani, aspergillus oryzae, yeast and the like.
The mannase is a hemicellulase, which degrades β -1,4 glycosidic bonds in an internal mode, mannose is at the non-reducing end of a degradation product, and action substrates of the mannase comprise glucomannan, galactomannan, β -mannan and the like, so that the mannase not only can reduce the intestinal viscosity and promote the digestion and absorption of nutrient substances, but also can eliminate the interference of β -mannan rich in beans on glucose absorption, greatly improve the energy digestion rate of cakes, especially bean pulp, and simultaneously improve the resistance and the uniformity of animals after the mannase is added.
The production of non-starch polysaccharase mainly adopts a biological fermentation method, and strains which can produce various non-starch polysaccharases and are searched by people at present comprise Trichoderma viride, Trichoderma reesei, Aspergillus niger, Penicillium funiculosum and the like, wherein the microorganisms for producing cellulase are mostly studied by fungi, and bacteria and actinomycetes, and the microorganisms which are currently used for producing cellulase are mainly Trichoderma, Aspergillus niger, Penicillium and Rhizopus.
The main energy feed resources in China are in short supply, but the application of grains and byproducts thereof in the feed is greatly limited by non-starch polysaccharide, so that the development of high-yield non-starch polysaccharide enzyme strains aiming at different feed backgrounds is urgently needed, the cost of applying the non-starch polysaccharide enzyme in the feed industry is reduced, and the problem of resource shortage is effectively relieved.
Disclosure of Invention
In view of the above, the invention provides a trichoderma reesei mutant strain capable of producing non-starch polysaccharidase in high yield and application thereof. The invention obtains the trichoderma reesei mutant strain with high yield of the non-starch polysaccharase by an ultraviolet mutagenesis method, can greatly improve the expression quantity of the non-starch polysaccharase, and can be widely applied to the production of the non-starch polysaccharase.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Trichoderma reesei strain with a preservation number of CCTCC NO: M2016363.
The xylanase activity in the mutant strain shake-flask fermentation supernatant is 102u/ml, is improved by 126.6% compared with the original strain, the cellulase activity is 95u/ml, is improved by 66.6% compared with the original strain, the β -glucanase activity is 229u/ml, is improved by 54.7% compared with the original strain, the mannanase activity is 25u/ml, is improved by 257.1% compared with the original strain, lactose is taken as an inducer, fermentation results of a fermentation tank show that the xylanase activity in the mutant strain trichoderma reesei NSP-51 fermentation supernatant is 1497u/ml, is improved by 305.6% compared with the original strain, the cellulase activity is 935/ml, is improved by 54% compared with the original strain, the xylanase activity is 1288u/ml, is improved by 56.5% compared with the original strain, the mannanase activity is 89u/ml, is improved by 323.8% compared with the original strain, liquid sugar is taken as the inducer, fermentation results of the fermentation tank show that the xylanase activity in the mutant strain trichoderma reesei NSP-51 fermentation supernatant is 206/ml, the preservation ratio is improved by 206/ml, the preservation ratio of the original strain is improved by 897.7.7.7.7%, the typical cellulase, the index of the mutant strain CGTakayama strain, the CGTakayase culture center is improved by 2016/363/70.7, the index of the CGTakayase, the CGTakayase culture center, the CGTakayase of the CGTakayase, the CGTakayase center, the CG.
The invention also provides application of the trichoderma reesei in fermentation production of non-starch polysaccharidase.
In some embodiments of the invention, the non-starch polysaccharide enzyme is a mixture of one or more of xylanase, cellulase, β -glucanase and mannanase.
The invention also provides a fermentation method for producing the non-starch polysaccharide enzyme, and the trichoderma reesei is taken as a fermentation strain.
In some embodiments of the invention, the fermentation process comprises shake flask fermentation and fermentor fermentation;
the fermentation medium of the fermentation method comprises: 2 parts of glucose; 1.5 parts of corn steep liquor; 0.9 part by mass of ammonium sulfate; 2 parts by mass of monopotassium phosphate; 0.4 part by mass of diammonium hydrogen phosphate; 0.15 part by mass of magnesium sulfate heptahydrate; 0.073 parts by mass of citric acid; 0.12 part by mass of calcium chloride; 0.075 part by mass of ferrous sulfate heptahydrate; 0.006 part by mass of zinc sulfate heptahydrate; 0.0012 part by mass of blue vitriol; 0.00053 part by mass of manganese sulfate monohydrate; boric acid 0.0003 part by mass.
In some embodiments of the present invention, the fermentation temperature of the shake flask fermentation in the fermentation method is 30 ℃, and the fermentation time is 5-7 days.
In some embodiments of the invention, the fermentor fermentation in the fermentation process comprises a shake flask culture and a fermentor culture;
the culture medium for shake flask culture comprises 10-30g/L of glucose and 200g/L of potato;
the shake flask culture condition is shake culture at 30 ℃ and 200rpm for 48 h.
In some embodiments of the invention, the conditions under which the fermentor is cultured in the fermentation process are: inoculating the fermentation strain into the fermentation culture medium for 24 hours at the temperature of 30 +/-1 ℃, the pH value of 5.0 +/-0.1 and the stirring speed of 600rpm, supplementing an inducer, controlling the dissolved oxygen to be 30-40%, and fermenting for 170 hours.
In some embodiments of the invention, the inducer in the fermentation process is lactose or liquid sugar.
The invention also provides non-starch polysaccharide enzyme obtained by fermentation by the fermentation method, wherein the non-starch polysaccharide enzyme is one or a mixture of more than two of xylanase, cellulase, β -glucanase and mannanase, and the ratio of the xylanase to the cellulase is 1: 2-3: 2.
The shake flask fermentation culture result shows that xylanase activity in the supernatant obtained by fermenting trichoderma reesei U1 serving as a starting strain is 45U/ml, cellulase activity is 57U/ml, β -glucanase activity is 148U/ml, and mannanase activity is 7U/ml, xylanase activity in the supernatant obtained by fermenting the mutant strain is 102U/ml and is increased by 126.6% compared with the starting strain, cellulase activity is 95U/ml and is increased by 66.6% compared with the starting strain, β -glucanase activity is 229U/ml and is increased by 54.7% compared with the starting strain, mannanase activity is 25U/ml and is increased by 257.1% compared with the starting strain, and unexpected technical effect is achieved.
The fermentation result of a fermentation tank shows that the xylanase activity in the supernatant obtained by fermenting trichoderma reesei U1 serving as an inducer is 369U/ml, the cellulase activity is 607U/ml, the β -glucanase activity is 823U/ml and the mannanase activity is 21U/ml, the xylanase activity in the supernatant obtained by fermenting trichoderma reesei NSP-51 serving as a mutant is 1497U/ml which is 305.6 percent higher than that of the original strain, the cellulase activity is 935U/ml which is 54 percent higher than that of the original strain, the xylanase activity of β -glucanase is 1288U/ml which is 56.5 percent higher than that of the original strain, and the mannanase activity is 89U/ml which is 323.8 percent higher than that of the original strain, so that unexpected technical effects are obtained.
Liquid sugar is used as an inducer, fermentation results of a fermentation tank show that the xylanase activity in the supernatant obtained by fermenting trichoderma reesei U1 serving as an initial strain is 16U/ml, the cellulase activity is 452U/ml, the β -glucanase activity is 599U/ml, and the mannanase activity is 13U/ml, the xylanase activity in the supernatant obtained by fermenting trichoderma reesei NSP-51 serving as a mutant strain is 206U/ml which is 1187% higher than that of the initial strain, the cellulase activity is 894U/ml which is 97.7% higher than that of the initial strain, the xylanase activity is β -glucanase activity is 925U/ml which is 54.4% higher than that of the initial strain, the mannanase activity is 34U/ml which is 161.5% higher than that of the initial strain, and unexpected technical effects are achieved.
Biological preservation Instructions
Biomaterial trichoderma reesei NSP-51, taxonomic name: trichoderma reesei NSP-51(Trichoderma reesei NSP-51), deposited at the China center for type culture Collection, Wuhan university, Wuhan, China, 2016, 7, 4, with the following addresses: wuhan university school in Wuchang Lojia mountain of Hubei province; the preservation number is CCTCC NO: M2016363.
Detailed Description
The invention discloses a trichoderma reesei mutant strain and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the strain as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention relates to a mutant strain Trichoderma reesei NSP-51(Trichoderma reesei NSP-51), which is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 2016, 7, 4 days, and the preservation number is CCTCC NO: M2016363.
The invention also relates to a novel non-starch polysaccharidase mixture produced by Trichoderma reesei with the preservation number of CCTCC NO: M2016363.
The invention provides a non-starch polysaccharide enzyme mixture which is produced by trichoderma reesei with the preservation number of CCTCC NO: M2016363, and at least comprises xylanase, cellulase, β -glucanase and mannanase.
In the non-starch polysaccharide enzyme mixture, the ratio of xylanase to cellulase is 1: 2-3: 2.
A method for producing non-starch polysaccharidase is prepared by fermenting the above Trichoderma reesei mutant strain.
The inducer used in the fermentation process is lactose or liquid sugar, and lactose is further preferable.
The components and mass ratio of the components of the culture medium used in the fermentation process are 2 percent of lactose; 1% of glucose; 1.5% of corn steep liquor; 0.9 percent of ammonium sulfate; 2% of monopotassium phosphate; diammonium phosphate 0.4%; magnesium sulfate heptahydrate 0.15%; 0.073% of citric acid; 0.12 percent of calcium chloride; ferrous sulfate heptahydrate 0.075%; 0.006% of zinc sulfate heptahydrate; 0.0012 percent of blue vitriol; 0.00053% of manganese sulfate monohydrate; boric acid 0.0003%.
After the mutant strain Trichoderma reesei NSP-51 obtained by screening is subjected to shake-flask fermentation for 5 days, the xylanase activity in a fermentation supernatant is 102U/ml and is increased by 126.6 percent compared with the original strain, the cellulase activity is 95U/ml and is increased by 66.6 percent compared with the original strain, β -glucanase activity is 229U/ml and is increased by 54.7 percent compared with the original strain, mannase activity is 25U/ml and is increased by 257.1 percent compared with the original strain, further, the applicant verifies through fermentation in a 20L fermentation tank, when lactose is taken as an inducer, the xylanase activity in the fermentation supernatant is 1497U/ml and is increased by 305.6 percent compared with the original strain after fermentation for 160h, the xylanase activity is increased by 54 percent compared with the original strain, the mannase activity is 1288U/ml and is increased by 56.5 percent compared with the original strain, the mannase activity is 89U/ml and is increased by 323 percent compared with the original strain, the mutant strain with liquid sugar, the mutant strain with the mutant strain NSP-51, the cellulase activity is capable of improving the cellulase, the cellulase activity of the cellulase, the cellulase is increased by 36, the cellulase is increased by about 2, the cellulase activity of the cellulase in the production of the cellulase, the cellulase in the cellulase, the cellulase is increased by the cellulase, the.
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 CURRENTPROTOLS 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 Trichoderma reesei mutant strain provided by the invention and raw materials and reagents used in the application thereof can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 starting bacterium Trichoderma reesei Shake flask fermentation and enzyme Activity detection
The original strain Trichoderma reesei U1(Trichoderma reesei U1) (the strain is selected from Laoshan soil in Qingdao city in 2015 at 2 months by Wujiapeng, one of the inventors) is inoculated to a fresh PDA plate and cultured for 5-7 days at 30 ℃.
A2 cm multiplied by 2 cm-sized bacterium block is cut and inoculated into 50ml of liquid shake flask culture medium (lactose 2%, glucose 1%, corn steep liquor 1.5%, ammonium sulfate 0.9%, potassium dihydrogen phosphate 2%, diammonium hydrogen phosphate 0.4%, magnesium sulfate heptahydrate 0.15%, citric acid 0.073%, calcium chloride 0.12%, ferrous sulfate heptahydrate 0.075%, zinc sulfate heptahydrate 0.006%, copper sulfate pentahydrate 0.0012%, manganese sulfate monohydrate 0.00053%, and boric acid 0.0003%) for fermentation, the mixture is cultured at 30 ℃ for 2 days, then the mixture is cultured at 25 ℃ for 3 days, after 5 days of culture, the fermentation broth is centrifuged, the obtained supernatant is crude enzyme liquid, and the fermentation supernatant is respectively subjected to cellulase, glucanase, xylanase and mannanase activity determination.
1. Cellulase activity detection
(1) Definition of the enzymatic Activity units of cellulases
The amount of enzyme required to release 1umol of reducing sugar in equivalent amounts of glucose per minute from a 5mg/ml solution of sodium carboxymethylcellulose degraded at 50 ℃ and a pH of 4.80 (neutral pH6.0) was one activity unit (IU).
(2) Enzyme activity measuring method
(2.1) drawing of standard curve:
and adding related test solutions into 8 test tubes according to the following table, adding 1.5ml of DNS reagent, fully shaking up, and reacting in a boiling water bath for 5 min. Rapidly cooling to room temperature, diluting to 5.0ml with water, and measuring absorbance of other test tube solutions at 540nm wavelength with test tube solution No. 0 as control. A standard curve was plotted with the absorbance as the ordinate and (glucose content/100) as the abscissa.
Test tube number 0 1 2 3 4 5 6 7
Buffer addition (ul) 1000 990 985 980 975 970 965 960
Glucose Standard solution addition amount (ul) 0 10 15 20 25 30 35 40
Glucose content (ug) 0 100 150 200 250 300 350 400
(2.2) enzyme activity determination:
adding 0.5ml CMC substrate into each of four test tubes, preheating with enzyme solution to be tested in 50 deg.C water bath for 5min, wherein the first three test tubes are sample test tubes, and the fourth test tube is blank tube. 0.5ml of the solution to be detected is added into each of the first three test tubes, and the reaction is carried out for 15min in a water bath at 50 ℃ while timing.
After the reaction, 1.5ml of DNS reagent was added to each of the first three tubes. Then, 1.5ml of LDNS is added into each blank tube in sequence, and finally, 0.5ml of enzyme solution to be detected is added into the blank tubes in sequence.
The three tubes were taken out and shaken up, and then reacted for 5min in a boiling water bath. It was rapidly cooled to room temperature and adjusted to 5.0ml with water. And (3) measuring the absorbance of the sample tube test solution under the condition of 540nm wavelength by taking a blank test tube test solution as a reference, wherein the absorbance is preferably between 0.25 and 0.30. If not, the dilution factor is changed and the re-measurement is carried out.
The enzyme activity calculation formula is as follows:
enzyme activity (IU/ml or IU/g) ═ glucose equivalent/180/15/0.5). times.n
In the formula: 180-conversion of glucose from microgram to micromole
15-reaction time of the test solution with the substrate
0.5-amount of enzyme to be measured added to the reaction
n-dilution factor of enzyme sample
2, β -glucanase Activity assay
(1) β definition of dextranase enzyme Activity Unit
The amount of enzyme required to release 1. mu. mol of reducing sugar by degradation from β -glucan solution at a concentration of 4mg/ml per minute at 37 ℃ and a pH of 5.5 was one enzyme activity unit U.
(2) Enzyme activity measuring method
(2.1) drawing of standard curve:
4.0ml of acetic acid-sodium acetate buffer solution is sucked, 5.0ml of DNS reagent is added, and the mixture is heated in boiling water bath for 5 min. Cooling to room temperature with tap water, and diluting to 25.0ml with water to obtain standard blank. Respectively sucking 1.00ml, 2.00ml, 3.00ml, 4.00ml, 5.00ml, 6.00ml and 7.00ml of glucose solution, respectively using acetic acid-sodium acetate buffer solution to make the volume to 100ml, and preparing into glucose standard solutions with the concentrations of 0.10mg/ml, 0.20mg/ml, 0.30mg/ml, 0.40mg/ml, 0.50mg/ml, 0.60mg/ml and 0.70 mg/ml.
Respectively sucking 2.00ml of the glucose standard solution with the concentration series (making two parallel solutions), respectively adding the glucose standard solutions into a graduated test tube, and respectively adding 2.0ml of acetic acid-sodium acetate buffer solution and 5.0ml of DNS reagent. Electromagnetically oscillating for 3-5 s, and heating in boiling water bath for 5 min. Then, the mixture was cooled to room temperature with tap water and made up to 25ml with water. The absorbance OD was measured at 540nm, with a standard blank as a control, adjusted to zero.
And drawing a standard curve by taking the glucose concentration as an axis Y and the absorbance OD value as an axis X. The standard curve needs to be redrawn each time a DNS reagent is newly formulated.
(2.2) enzyme activity determination:
10.0ml of β -dextran solution was aspirated and equilibrated at 37 ℃ for 20 min.
10.0ml of the appropriately diluted enzyme solution was aspirated and equilibrated at 37 ℃ for 10 min.
Absorbing 2.00ml of enzyme solution (which is balanced at 37 ℃), adding the enzyme solution into a graduated test tube, adding 5ml of DNS reagent, electromagnetically oscillating for 3s, then adding 2.0ml of β -dextran solution, balancing for 30min at 37 ℃, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, adding water to fix the volume to 25ml, electromagnetically oscillating for 3 s-5 s, taking a standard blank sample as a blank control, and measuring the absorbance A at 540nmB。
2.00ml of the appropriately diluted enzyme solution (equilibrated at 37 ℃) was aspirated and added to a graduated tube, followed by 2.0ml of β -dextran solution(after 37 ℃ equilibrium), electromagnetically oscillating for 3s, and accurately preserving the temperature for 30min at 37 ℃. 5.0ml of DNS reagent was added and the reaction was magnetically shaken for 3 seconds to stop the enzymatic reaction. Heating in boiling water bath for 5min, cooling to room temperature with tap water, adding water to desired volume of 25ml, and electromagnetically oscillating for 3 s. Measuring absorbance A at 540nm with standard blank as blankE。
The enzyme activity calculation formula is as follows:
Figure BDA0001083045990000091
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
3. Xylanase activity assay
(1) Definition of xylanase Activity units
The enzyme amount required for releasing 1 mu mol of reducing sugar from 5mg/ml xylan solution per minute at 37 ℃ and pH5.5 is an enzyme activity unit U.
(2) Enzyme activity measuring method
Taking 2ml of xylan substrate with the concentration of 1% (prepared by a pH5.5 acetic acid-sodium acetate buffer solution), adding the xylan substrate into a colorimetric tube, balancing for 10min at 37 ℃, adding 2ml of acidic xylanase enzyme solution which is properly diluted by the pH5.5 acetic acid-sodium acetate buffer solution and well balanced at 37 ℃, uniformly mixing, and accurately preserving the temperature at 37 ℃ for reaction for 30 min. After the reaction was completed, 5ml of DNS reagent was added and mixed well to terminate the reaction. Boiling in boiling water bath for 5min, cooling to room temperature with tap water, adding distilled water to constant volume to 25ml, mixing, measuring absorbance A at 540nm with standard blank as blank controlE。
The enzyme activity calculation formula is as follows:
Figure BDA0001083045990000101
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
4. Mannanase activity assay
(1) Definition of the mannanase enzyme Activity Unit
The amount of enzyme required to release 1umol of reducing sugar by degradation per minute from a mannan solution having a concentration of 3mg/ml at 37 ℃ and a pH of 5.5 was one enzyme activity unit U.
(2) Enzyme activity measuring method
(2.1) drawing of standard curve:
4.0ml of acetic acid-sodium acetate buffer solution is sucked, 5.0ml of DNS reagent is added, and the mixture is heated in boiling water bath for 5 min. Cooling to room temperature with tap water, and diluting to 25.0ml with water to obtain standard blank.
Respectively sucking 1.00, 2.00, 3.00, 4.00, 5.00, 6.00 and 7.00ml of mannose solution (5.5), respectively using acetic acid-sodium acetate buffer solution to fix the volume to 100ml, and preparing the D-mannose standard solution with the concentration of 0.10-0.70 mg/ml.
Respectively sucking 2.00ml of mannose standard solution of the concentration series (making two parallel solutions), respectively adding into a graduated test tube, and respectively adding 2ml of acetic acid-sodium acetate buffer solution and 5ml of DNS reagent. Electromagnetically oscillating for 3s, and heating in boiling water bath for 5 min. Then, the mixture was cooled to room temperature with tap water and made up to 25ml with water. The absorbance OD was measured at 540nm, with a standard blank as a control, adjusted to zero.
And drawing a standard curve by taking the mannose concentration as an axis Y and the absorbance OD value as an axis X. The standard curve needs to be redrawn each time a DNS reagent is newly formulated.
(2.2) enzyme activity determination:
10.0ml of mannan solution was aspirated and equilibrated at 37 ℃ for 10 min.
10.0ml of the appropriately diluted enzyme solution was aspirated and equilibrated at 37 ℃ for 10 min.
2.00ml of the enzyme solution (equilibrated at 37 ℃) diluted appropriately is aspirated, added to a graduated tube, and 5ml of DNS reagent is added thereto, and subjected to electromagnetic oscillation for 3 seconds. Then adding 2.0ml mannan solution, keeping the temperature at 37 ℃ for 30min, and heating in boiling water bath for 5 min. Cooling to room temperature with tap water, adding water to a constant volume of 25ml, and electromagnetically oscillating for 3 s. Measuring absorbance A at 540nm with standard blank as blankB。
2.0ml of the enzyme solution (equilibrated at 37 ℃) diluted appropriately is aspirated, added to a graduated test tube, and then 2.0ml of mannan solution (equilibrated at 37 ℃) is added, electromagnetically shaken for 3 seconds, and precisely incubated at 37 ℃ for 30 min. Adding 5.0ml DNS reagent, electromagnetically oscillating for 3s, and performing enzymolysis reaction. Heating in boiling water bath for 5min, cooling to room temperature with tap water, adding water to desired volume of 25ml, and electromagnetically oscillating for 3 s. Measuring absorbance A at 540nm with standard blank as blankE。
The enzyme activity calculation formula is as follows:
Figure BDA0001083045990000111
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
Preparation method of 5 liquid sugar
500g of glucose was weighed into a 1L beaker, distilled water was added to about 900mL, after all was dissolved, 25mL of 85% phosphoric acid was added, and then the volume was adjusted to 1L, and the mixture was treated in a sterilizer at 121 ℃ for 30 min. The brown solution obtained is liquid sugar.
Example 2 UV mutagenesis and mutant Strain screening
Determination of the lethality rate: inoculating original strain Trichoderma reesei U1 to PDA plate, and culturing at 30 deg.C for 5-7 d. Surface of bacterial colonyWhen a large amount of spores are produced, 5ml of sterile water is sucked for elution to obtain a spore liquid, the spore liquid is resuspended in sterile water after centrifugation, and the spore liquid is counted by a hemocytometer to make the spore concentration about 5X 107One per ml. A90 mm sterile petri dish placed in a rotor was added with 10ml of diluted spore suspension, and stirred 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 60s, 90s, 120s, 150s and 180s, respectively, diluting the irradiated spore solution by 10000 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 90% when the irradiation time is 120s, and the irradiation time is selected for subsequent mutagenesis experiments.
Mutagenesis screening: a90 mm sterile petri dish placed in a rotor was added with 10ml of diluted spore suspension (concentration 5X 10)7) 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 120s, standing in dark for 30min, diluting by 10000 times, coating 100ul PDA plate, and culturing at 30 deg.C for 2-3 d.
Totally coating 150 PDA plates, culturing at 30 ℃ for 2-3 days, allowing each plate to grow about 50 colonies, observing colony morphology, selecting 200 mutants with small colony morphology and dense mycelia, inoculating to the PDA plates, and culturing at 30 ℃ for 5-7 days. Selecting 150 colonies with normal growth state, cutting 2cm multiplied by 2cm fungus blocks of each mutant colony, respectively inoculating the colonies in 50ml liquid shake flask culture medium (lactose 2%, glucose 1%, corn steep liquor 1.5%, ammonium sulfate 0.9%, potassium dihydrogen phosphate 2%, diammonium hydrogen phosphate 0.4%, magnesium sulfate heptahydrate 0.15%, citric acid 0.073%, calcium chloride 0.12%, ferrous sulfate heptahydrate 0.075%, zinc sulfate heptahydrate 0.006%, copper sulfate pentahydrate 0.0012%, manganese sulfate monohydrate 0.00053%, boric acid 0.0003%), fermenting, culturing at 30 ℃ for 2 days, and then culturing at 25 ℃ for 3 days; centrifuging the thallus to obtain supernatant as crude enzyme solution. And finally screening a mutant strain with the highest NSP enzyme yield by carrying out NSP enzyme activity detection on the obtained crude enzyme solution, and naming the mutant strain as Trichoderma reesei NSP-51(Trichoderma reesei NSP-51).
The fermentation result is shown in Table 1. the xylanase activity in the mutant strain fermentation supernatant is 102u/ml, which is increased by 126.6% compared with the original strain, the cellulase activity is 95u/ml, which is increased by 66.6% compared with the original strain, the enzyme activity of β -glucanase is 229u/ml, which is increased by 54.7% compared with the original strain, and the mannanase activity is 25u/ml, which is increased by 257.1% compared with the original strain, thereby obtaining unexpected technical effects.
TABLE 1
Figure BDA0001083045990000121
The applicant has deposited the above mutant strain Trichoderma reesei NSP-51(Trichoderma reesei NSP-51) in the China center for type culture Collection, CCTCC NO: M2016363, Wuhan university, Wuhan, China at 2016, 7, 4.
Example 320L fermenter fermentation validation
1. Lactose as inducer
Inoculating original strain Trichoderma reesei U1 and mutant strain Trichoderma reesei NSP-51 to the same shake flask seed culture medium (glucose 10-30g/L, potato 100 g/L) respectively, carrying out shake cultivation at 30 ℃ and 200rpm for 48h, then transferring the fermentation broth into a 20L fermentation tank (the formula is glucose 2%, corn steep liquor 1.5%, ammonium sulfate 0.9%, potassium dihydrogen phosphate 2%, diammonium hydrogen phosphate 0.4%, magnesium sulfate heptahydrate 0.15%, citric acid 0.073%, calcium chloride 0.12%, ferrous sulfate heptahydrate 0.075%, zinc sulfate heptahydrate 0.006%, copper sulfate pentahydrate 0.0012%, manganese sulfate monohydrate 0.00053%, and boric acid 0.0003%), controlling the temperature at 30 + -1 ℃, controlling the pH value at 5.0 + -0.1, controlling the stirring speed at 600rpm, after culturing in the fermentation tank for 24h, beginning to supplement lactose for inducing the production of thallus, controlling the dissolved oxygen at 30-40%, the fermentation time is about 170h, and zymocyte liquid is prepared.
The zymophyte liquid is centrifuged, and the supernatant is taken, the result is shown in a table 2. the xylanase activity in the supernatant obtained by fermenting trichoderma reesei U1 serving as the starting bacterium is 369U/ml, the cellulase activity is 607U/ml, the β -glucanase activity is 823U/ml, and the mannanase activity is 21U/ml. the xylanase activity in the supernatant obtained by fermenting trichoderma reesei NSP-51 serving as the mutant bacterium is 1497U/ml which is improved by 305.6 percent compared with the starting bacterium, the cellulase activity is 935U/ml which is improved by 54 percent compared with the starting bacterium, the enzyme activity of β -glucanase is 1288U/ml which is improved by 56.5 percent compared with the starting bacterium, and the mannanase activity is 89U/ml which is improved by 323.8 percent compared with the starting bacterium, so that unexpected technical effects are obtained.
TABLE 2
Figure BDA0001083045990000131
2. Liquid sugar as inducer
Inoculating original strain Trichoderma reesei U1 and mutant strain Trichoderma reesei NSP-51 to the same shake flask seed culture medium (glucose 10-30g/L, potato 100 g/L) respectively, carrying out shake culture at 30 ℃ and 200rpm for 48h, then transferring the fermentation broth into a 20L fermentation tank (the formula is glucose 2%, corn steep liquor 1.5%, ammonium sulfate 0.9%, potassium dihydrogen phosphate 2%, diammonium hydrogen phosphate 0.4%, magnesium sulfate heptahydrate 0.15%, citric acid 0.073%, calcium chloride 0.12%, ferrous sulfate heptahydrate 0.075%, zinc sulfate heptahydrate 0.006%, copper sulfate pentahydrate 0.0012%, manganese sulfate monohydrate 0.00053%, and boric acid 0.0003%), controlling the temperature at 30 + -1 ℃, controlling the pH value at 5.0 + -0.1, controlling the stirring speed at 600rpm, after culturing in the fermentation tank for 24h, beginning to supplement liquid sugar to induce thallus to produce enzyme, controlling the dissolved oxygen at 30-40%, the fermentation time is about 170h, and zymocyte liquid is prepared.
The zymophyte liquid is centrifuged, and the supernatant is taken, the result is shown in a table 3. the xylanase activity in the supernatant obtained by fermenting the original strain trichoderma reesei U1 is 16U/ml, the cellulase activity is 452U/ml, the β -glucanase activity is 599U/ml, and the mannanase activity is 13U/ml, the xylanase activity in the supernatant obtained by fermenting the mutant strain trichoderma reesei NSP-51 is 206U/ml which is 1187 percent higher than that of the original strain, the cellulase activity is 894U/ml which is 97.7 percent higher than that of the original strain, the xylanase activity is β -glucanase activity is 925U/ml which is 54.4 percent higher than that of the original strain, and the mannanase activity is 34U/ml which is 161.5 percent higher than that of the original strain, so as to obtain unexpected technical effects.
TABLE 3
Figure BDA0001083045990000141
Compared with fermentation enzyme activity data of a fermentation liquid taking lactose and liquid sugar as inducers, the expression quantity of each component of non-starch polysaccharide enzyme in the mutant strain Trichoderma reesei NSP-51 is improved by more than 50%, the inhibition effect of glucose on xylanase expression is mainly reduced, the ratio of xylanase is greatly improved, the enzyme activity ratio of xylanase and cellulase in the fermentation liquid is improved from about 1:2 to about 3:2 under the induction of lactose, and unexpected effects are obtained.
In conclusion, the mutant strain Trichoderma reesei NSP-51 obtained by combining the mutagenesis technology with the shake flask screening can greatly improve the yield of the non-starch polysaccharide enzyme, can obviously improve the expression proportion of xylanase, is favorable for reducing the production cost, and has wide application prospect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (8)

1. Trichoderma reesei (T. reesei) ((T. reesei))Trichoderma reesei) The preservation number is CCTCC NO: M2016363.
2. Use of trichoderma reesei according to claim 1 for the fermentative production of non-starch polysaccharidases.
3. The use according to claim 2, wherein the non-starch polysaccharide enzyme is one or a mixture of two of xylanase, cellulase, β -glucanase and mannanase.
4. A fermentation process for producing a non-starch polysaccharide enzyme, wherein the Trichoderma reesei strain according to claim 1 is used as a fermentation strain.
5. The fermentation process of claim 4, wherein the fermentation process comprises shake flask fermentation and fermentor fermentation;
the fermentation medium of the fermentation method comprises: 2 parts of glucose; 1.5 parts of corn steep liquor; 0.9 part by mass of ammonium sulfate; 2 parts by mass of monopotassium phosphate; 0.4 part by mass of diammonium hydrogen phosphate; 0.15 part by mass of magnesium sulfate heptahydrate; 0.073 parts by mass of citric acid; 0.12 part by mass of calcium chloride; 0.075 part by mass of ferrous sulfate heptahydrate; 0.006 part by mass of zinc sulfate heptahydrate; 0.0012 part by mass of blue vitriol; 0.00053 part by mass of manganese sulfate monohydrate; boric acid 0.0003 part by mass.
6. The fermentation method according to claim 5, wherein the shake flask fermentation is carried out at a fermentation temperature of 30 ℃ for 5-7 days.
7. The fermentation process of claim 5, wherein the conditions of the fermentor culture are: inoculating fermentation strain into the fermentation medium of claim 5 at 30 + -1 deg.C, pH 5.0 + -0.1, stirring at 600rpm, culturing for 24 hr, adding inducer, controlling dissolved oxygen at 30-40%, and fermenting for 170 hr.
8. The fermentation process of claim 7, wherein the inducer is lactose or liquid sugar.
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