CN117586999A - New semi-continuous fermentation high-yield enzyme process - Google Patents
New semi-continuous fermentation high-yield enzyme process Download PDFInfo
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- CN117586999A CN117586999A CN202210970410.3A CN202210970410A CN117586999A CN 117586999 A CN117586999 A CN 117586999A CN 202210970410 A CN202210970410 A CN 202210970410A CN 117586999 A CN117586999 A CN 117586999A
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the technical field of protein expression, in particular to a novel process for semi-continuous fermentation and high enzyme production. In the invention, mixed sugar of fed-batch glucose and lactose is used as an inducer to induce the expression of maltooligosaccharide-based trehalose synthase or maltooligosaccharide-based trehalose hydrolase in recombinant bacteria, wherein the recombinant bacteria contain optimized coding genes, and the nucleotide sequence of the coding genes is shown as SEQ ID NO.1 or SEQ ID NO. 2. The invention can control the growth of thalli and the expression of protein simultaneously by adopting a glucose and lactose mixed sugar feeding process; in addition, part of fermentation liquid is discharged in the fermentation process, and part of fresh fermentation medium is supplemented, so that the inhibition effect of acetic acid on the growth of thalli and the expression of protein can be reduced, the maintenance time of the vitality of thalli is prolonged, and the total thalli amount and the expression amount of protein are increased.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a novel semi-continuous fermentation high-yield enzyme process.
Background
Trehalose is a non-reducing disaccharide formed by condensing two glucopyranose molecules through alpha-1, 1 glycosidic bonds, and is divided into crystalline trehalose and anhydrous trehalose according to the presence or absence of crystalline moisture. Currently commercial trehalose is mainly crystalline trehalose, contains two crystal waters, and has a molecular formula of C 12 H 22 O 11 ·2H 2 O, molecular weight 378.33. Trehalose has a reputation for "vital sugars" in the scientific community, due to its characteristic of protecting biological activity under extreme conditions. Besides the protection function for organisms, the trehalose also has the functions of preventing starch from aging, preventing protein from denaturation, inhibiting lipid from oxidative deterioration, inhibiting the generation of fishy smell, being beneficial to the preservation of fruits and vegetables, and the like, so that the trehalose is widely applied to the industries of foods, medicines and cosmetics.
The current production method of trehalose mainly comprises a microorganism extraction method, a fermentation method, an enzyme conversion method and a gene recombination method. The enzyme method for synthesizing the trehalose mainly comprises four ways, including a trehalose-6-phosphate synthase and trehalose-6-phosphatase conversion method, a trehalose phosphorylase conversion method, a trehalose synthase conversion method (single enzyme method) and a maltooligose trehalose synthase (MTse) and maltooligose trehalose hydrolase (MTHase) conversion method (double enzyme method), wherein the former two methods have higher cost and unstable phosphorylase due to the need of high-energy compounds as raw materials, and are not suitable for industrial production; the latter two methods have simple conversion process operation, high conversion rate and low cost, and are the main way for industrially producing trehalose at present.
Trehalose synthase, MTse, MTHase and the like are mainly derived from wild fatty bacillus, pseudomonas putida, thermophilic aquatic bacteria, corynebacterium glutamicum, micrococcus roseus and sulfolobus solfataricus, but the enzyme activity of the obtained enzyme solution is low, the separation is difficult and the conversion efficiency is low due to low enzyme yield and high impurity protein content of the wild bacteria. Along with the development of modern genetic engineering technology, in order to improve the conversion efficiency of starch to trehalose and reduce the production cost, the use of genetic engineering bacteria for heterologous efficient expression of target proteins has become a current research hotspot. The heterologous protein expression systems commonly used today include prokaryotic protein expression systems (e.g., colibacillus protein expression systems and bacillus subtilis protein expression systems) and eukaryotic protein expression systems (e.g., mammalian protein expression systems and yeast protein expression systems).
Jiang Nada discloses a genetically engineered bacterium for high yield of MTse in patent CN106190880A, which uses pPIC3.5k as a vector, uses Pichia pastoris KM71 as an expression host, expresses MTse from sulfolobus acidocaldarius, and ferments to produce the MTse with the enzyme activity reaching 3128.7U/mL. Meanwhile, in a patent CN105969713A, a genetic engineering bacterium for high yield of MTHase is disclosed, pET-32a (+) is used as a carrier, E.coli Origami (DE 3) is used as an expression host, the genetic engineering bacterium for high yield of MTHase is constructed, and the enzyme activity of MTHase produced by fermenting the strain can reach 204U/mL. Patent CN109337851B discloses a method for efficiently displaying trehalose synthase on the spore surface of bacillus subtilis, in which TreS genes of trehalose synthase are fused with 5 encoding genes of spore capsid proteins respectively through genetic engineering means, and bacillus subtilis integrated plasmid is used as a carrier to be transformed into bacillus subtilis, so as to obtain genetic engineering bacteria capable of efficiently displaying trehalose synthase on the spore surface.
The colibacillus protein expression system is widely applied because of the advantages of high cell reproduction speed, high yield, low price, high expression level, strong anti-pollution capability, definite genetic background and the like, but the system is still affected by various factors in practical application, wherein the formation of inclusion bodies is a major problem which puzzles people. The main reason for inclusion body formation is that the foreign target protein is misfolded during expression due to the fact that the protein synthesis rate exceeds the protein folding rate, so that inactive insoluble substances are formed. In order to obtain soluble proteins, protein renaturation is required or it is desirable to express them in the supernatant in the course of designing experiments. The stability of the secondary structure of mRNA can be improved through codon optimization, so that the correct folding of a nascent peptide segment is facilitated, and the expression of exogenous active protein is improved. Or fusion proteins such as fusion expression molecular chaperones and the like are used for improving the expression quantity and the solubility of the exogenous proteins. Or the soluble expression of the recombinant protein is improved by optimizing the induction expression conditions, including reducing the induction temperature, reducing the protein synthesis rate, optimizing the induction culture conditions such as the concentration of the inducer, the induction time and the like.
Secondly, in order to obtain a large amount of target proteins, a fed-batch fermentation process is generally adopted to realize high-density culture, but because the oxygen demand is large in the escherichia coli fermentation culture process, the dissolved oxygen is insufficient to generate a large amount of acetic acid along with the increase of the volume in the high-density culture process, so that the growth of thalli and the expression of exogenous proteins are inhibited.
Microbial fermentation can be divided into various modes of batch, fed-batch, semi-continuous, etc. The consumption of manpower and material resources for batch fermentation is large, each batch of fermentation needs to be carried out such as loading, sterilization, inoculation, discharging, cleaning and the like, the procedures are complicated, the fermentation period is long, and the production efficiency is low; fed-batch fermentation can supplement the deficiency of nutrients or precursors by feeding, but the synthesis of the product is ultimately inevitably inhibited due to the continued accumulation of harmful metabolites; the continuous fermentation has greatly improved production strength compared with batch fermentation and fed-batch fermentation, but is easy to be polluted by mixed bacteria, the strain is easy to be degraded, the equipment investment is larger, and the concentration of fermentation products is lower; in the semi-continuous fermentation process, by discharging part of fermentation liquor and supplementing fresh culture medium, not only can nutrients and precursors be supplemented, but also metabolic harmful substances are diluted, thereby being beneficial to the continuous synthesis of products.
Disclosure of Invention
The purpose of the present invention is to increase the yield and the enzyme activity of maltooligosaccharide-based trehalose synthase or maltooligosaccharide-based trehalose hydrolase.
The method has a certain improvement effect on the soluble expression of the foreign protein by means of molecular biology such as codon optimization, but can not completely solve the problem that the protein synthesis rate is high in the actual induced expression process, so that misfolding forms inclusion bodies; the solubility of the foreign protein can be improved by coexpression of the fusion protein such as chaperone, etc., but a subsequent treatment step is required to be added, and the fusion protein is removed by an enzyme reaction to obtain the target protein. The temperature-induced method can prevent the formation of insoluble inclusion bodies of wrong protein intermediates generated by too fast folding rate, but too low temperature can lead to the reduction of nutrient uptake rate and growth rate, and the yield of target proteins can be lower; the fed-batch fermentation process can increase the bacterial body quantity and the total enzyme quantity, but with the increase of the fermentation volume, the insufficient supply can lead to the continuous accumulation of acetic acid, and finally inhibit the bacterial body growth and the protein expression.
In order to achieve the object of the present invention, the amount of cells is maintained large while the enzymatic activity of cells in a fermentation broth is high. In a first aspect, the present invention provides a semi-continuous fermentation process in which a mixed sugar of glucose and lactose is fed as an inducer to induce expression of maltooligosyl trehalose synthase or maltooligosyl trehalose hydrolase in recombinant bacteria.
In the semi-continuous fermentation method provided by the invention, the total sugar concentration of the mixed sugar is 40-80%; controlling dissolved oxygen value to be 30-40% in the fed-batch mixed sugar control fermentation system; preferably, the flow acceleration of the mixed sugar is 2-5g/L/h.
The total sugar concentration refers to the concentration of glucose and lactose, for example, 40% glucose concentration, 20% lactose concentration, and 60% total sugar concentration; in the semi-continuous fermentation method provided by the invention, the mass ratio of glucose to lactose is as follows: 1:1-3:1.
In the semi-continuous fermentation method provided by the invention, the recombinant bacteria contain the optimized coding gene of the maltooligosaccharide-based trehalose synthase, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.
In the semi-continuous fermentation method provided by the invention, the recombinant bacteria contain the optimized coding gene of maltooligosaccharide-based trehalose hydrolase, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
More specifically, in the semi-continuous fermentation method provided by the invention, semi-continuous fermentation is performed by feeding and feeding simultaneously, and the method comprises the following steps:
(1) Taking escherichia coli containing the coding gene shown in any one of SEQ ID NO.1 or SEQ ID NO.2 as engineering bacteria, and inoculating the activated escherichia coli seed liquid into a fermentation medium;
(2) Starting material discharge when the volume of fermentation liquor is 40-50% of the volume of the fermentation vessel, adding a fed-batch culture medium accounting for 40-60% of the material discharge amount at the same time when each material discharge is performed, and continuing feeding sugar to induce enzyme production;
the fed-batch culture medium contains glucose, hydrolysate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
(3) And (3) repeating the step (2) when the volume of the fermentation liquor reaches 40-50% again.
In the semi-continuous fermentation method provided by the invention, the induction culture in the step (2) comprises the following steps: inoculating the activated escherichia coli seed liquid into a fermentation culture medium, controlling the dissolved oxygen of a fermentation system to be 20% -30% before induction, and controlling the dissolved oxygen of the fermentation system to be 30% -40% by feeding sugar after the dissolved oxygen is fully consumed and the dissolved oxygen rises;
preferably, during the induction culture, the glucose content in the fermentation broth is zero, and the fermentation temperature is reduced by 30-33 ℃.
More specifically, in the semi-continuous fermentation method provided by the invention, in a semi-continuous fermentation culture system, the pH is controlled to be 6.9-7.1, the air quantity is controlled to be 10-30L/min, the rotating speed is controlled to be 200-700 rpm, and the tank pressure is controlled to be 0.05-0.1 Mpa.
The invention also claims the coding gene shown in any one of SEQ ID NO.1 or SEQ ID NO.2 or the application of the semicontinuous fermentation method in improving the yield or the enzyme activity of the recombinant maltooligosaccharide-producing trehalose synthase or maltooligosaccharide-producing trehalose hydrolase according to the understanding of the person skilled in the art.
And the application of the semi-continuous fermentation method in synthesizing the trehalose by a double enzyme method.
The level of enzyme activity depends on the level of soluble active protein, and increasing soluble expression reduces inclusion body formation and can increase the level of enzyme activity. According to the protein dynamics model research, the yield of active protein is determined by the protein synthesis rate, the protein folding rate and the protein aggregation rate. At high levels of expression, the rate of aggregation of nascent peptide chains once exceeded the rate of protein folding leads to inclusion body formation. Therefore, reducing the rate of recombinant protein synthesis is beneficial to improving the soluble expression of recombinant protein, and reducing the culture temperature can reduce the rate of recombinant protein synthesis, but at the same time, the rate of bacterial growth is reduced.
The expression level of the exogenous protein in the escherichia coli expression system mainly depends on the enzyme expression quantity and the enzyme activity level. Experiments prove that by adopting the semi-continuous fermentation method (process) provided by the application, the enzyme activity of the maltooligosaccharide-based trehalose synthase can be improved by 1.76-2.4 times, and the bacterial body quantity can be improved by 1.42-1.5625 times; the enzyme activity of the mycotic maltooligosyl trehalose hydrolase is improved by 1.2-1.525 times, and the mycotic quantity is improved by 1.75-1.94 times.
The invention has the beneficial effects that:
(1) The method adopts a stronger promoter in the strain construction stage, and selects a proper vector and host bacteria, so that the expression quantity of target protein can be improved; the coding gene shown in any one of SEQ ID NO.1 or SEQ ID NO.2 adopted by the invention has good expression capacity in an escherichia coli expression system.
(2) The glucose and lactose mixed sugar feeding process can control the growth of thalli and the expression of protein simultaneously; the invention can realize the soluble expression of the exogenous protein by controlling the sugar supplementing rate, and has simple operation; the invention adopts a semi-continuous fermentation process, has sufficient dissolved oxygen, avoids the accumulation of acetic acid, improves the culture environment of microorganisms, and is beneficial to keeping the stability of the activity of thalli.
(3) The invention adopts a semi-continuous fermentation process, does not need repeated operations of loading, sterilizing, inoculating, discharging and the like, saves non-fermentation time, has high equipment utilization rate and high total enzyme yield. The culture medium nitrogen source hydrolysate adopted by the invention has wide sources and low cost, and is beneficial to industrial application.
(4) The invention can increase the total expression amount of target protein by optimizing culture conditions in the fermentation culture process. The invention adopts a semi-continuous fermentation process, part of fermentation liquid is discharged at intervals in the fermentation process, and part of fresh fermentation culture medium is supplemented, so that the inhibition effect of acetic acid production on the growth of thalli and the expression of protein can be reduced, the maintenance time of the vitality of thalli is prolonged, and the total thalli quantity and the expression quantity of protein are increased.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The technology used in the invention, such as vector construction technology, engineering bacteria construction technology, electrophoresis technology and the like, is a relatively mature technology in genetic engineering, and can be realized by a person skilled in the art according to the prior art. The equipment or reagents, carriers, enzymes, etc. used in the operation are available in the market unless otherwise specified.
In the invention, the formula of the TB medium (W/V) is as follows: 1.2% peptone, 2.4% yeast powder, 0.213% potassium dihydrogen phosphate, 1.643% dipotassium hydrogen phosphate, 0.5% glycerol, pH7.0.
The preparation method of the PBS buffer solution comprises the following steps: 8.0g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 are weighed and dissolved in 800mL of distilled water, the pH of the solution is regulated to 6.0 by HCl, and finally distilled water is added to constant volume to 1L to obtain the nano-powder.
The method for crushing the thalli comprises the following steps: and (3) centrifuging at 10000rpm for 15min, collecting thalli in the fermentation broth, accurately weighing 1g thalli into a beaker, dissolving the thalli to a constant volume to 50mL by using PBS buffer (pH=6.0), crushing the bacterial suspension by using an ultrasonic cell crusher, wherein the total crushing time is 40min, the ultrasonic working time is 1s, the ultrasonic gap time is 2s and the ultrasonic power is 70% at intervals of 10 min.
The method for measuring the enzyme activity of the MTase-producing bacteria comprises the following steps: firstly absorbing 25uL of 1% maltohexaose solution, then absorbing 375uL of PBS buffer solution (pH=6.0), adding 100uL of MTse-producing bacterial body breaking solution, reacting for 30min in a water bath kettle at 50 ℃, then inactivating in a boiling water bath for 10min until the maltohexaose solution is completely cooled, and measuring the content of the maltohexaose after the reaction by adopting a high performance liquid chromatograph.
MTse enzyme activity was defined as: the amount of MTase enzyme required to convert 1umol maltohexaose per 1min was 1U.
The method for measuring the enzyme activity of the MTHase-producing bacteria comprises the following steps: 200uL of 9% maltodextrin solution is sucked, 500uL of PBS buffer solution (pH=6.0) is sucked, 100uL of MTSAE-producing thallus crushing liquid is added, the mixture is reacted in a water bath kettle at 55 ℃ for 60min and then put into a boiling water bath for inactivation for 10min, after the mixture is cooled, 100uL of MTHAE-producing thallus crushing liquid is added, and then the mixture is reacted in the water bath kettle at 55 ℃ for 20min, the reaction is stopped in the boiling water bath for inactivation for 10min, and the content of the trehalose in the reaction liquid is measured by adopting a high performance liquid chromatograph.
MTHase enzyme activity was defined as: the amount of MTHase enzyme required to convert maltodextrin to 1umol trehalose per 1min was 1U.
Measuring glucose in the fermentation liquid by adopting a biological Sensor (SBA);
trehalose determination the trehalose content was determined by high performance liquid chromatography in GB/T23529-2009.
EXAMPLE 1 construction of recombinant expression strains producing MTase and MTHase
This example provides the construction of recombinant expression strains of MTase and MTHase, comprising the following steps:
the gene treY is artificially synthesized according to the (maltooligosyl trehalose synthase) MTse gene sequence of Arthrobacter source reported on NCBI, the nucleotide sequence is shown as SEQ ID NO.1, the recombinant expression plasmid pET-28a-treY is obtained by connecting the expression vector pET-28a and treY gene after double enzyme digestion by adopting restriction enzymes, the recombinant expression plasmid pET-28a-treY is transformed into E.coli BL21 (DE 3) competent cells, the competent cells are cultured for 1h at 37 ℃, the competent cells are coated on a solid TB culture medium, and single colonies are picked up and cultured for 12h at 37 ℃ in a TB liquid culture medium containing kanamycin resistance for PCR verification. And (3) carrying out glycerol pipe preservation on the strain with correct verification to obtain a recombinant expression strain E.coli BL21 (DE 3)/pET-28 a-treY.
The gene treZ is artificially synthesized according to the (maltooligosyl trehalose hydrolase) MTHase gene sequence reported on NCBI, the nucleotide sequence is shown as SEQ ID NO.2, the recombinant expression plasmid pET-24a-treZ is obtained by connecting the expression vector pET-24a and treZ gene after double enzyme digestion by adopting restriction enzymes, the recombinant expression plasmid pET-24a-treZ is transformed into E.coli BL21 (DE 3) LysS competent cells, the E.coli BL21 (DE 3) LysS competent cells are cultured for 1h at 37 ℃, the E.coli BL 21) is coated on a solid TB flat plate, and single colonies are picked up and cultured for 12h at 37 ℃ in a liquid culture medium containing kanamycin resistance TB for PCR verification. And (3) carrying out glycerol pipe preservation on the strain with correct verification to obtain a recombinant expression strain E.coli BL21 (DE 3) LysS/pET-24a-treZ.
EXAMPLE 2 seed culture
This example provides the steps of culturing the recombinant bacterium obtained in example 1 as seeds, as follows:
activating and culturing recombinant expression strains E.coli BL21 (DE 3)/pET-28 a-treY and E.coli BL21 (DE 3) LysS/pET-24a-treZ respectively coated with kanamycin resistance TB flat plate at 37deg.C overnight, inoculating 3 rings into sterilized seed culture medium, inoculating 500mL triangular flask with liquid amount of 50mL, shaking culture at 37deg.C, and culturing at 220rpm to OD 600 Stopping culturing until the culture time reaches 8-10.
The seed culture medium consists of: glucose 20g/L, hydrolysate 20g/L, dipotassium hydrogen phosphate 5.5g/L and potassium dihydrogen phosphate 0.8g/L.
Example 3 semi-continuous fermentation enzyme production Process
The embodiment provides a semi-continuous fermentation process, which comprises the following steps:
(1) Preparing materials:
fermentation medium: 25g/L of glucose, 45g/L of hydrolysate, 8g/L of dipotassium hydrogen phosphate and 2g/L of monopotassium phosphate;
feeding a culture medium: 90g/L of hydrolysate, 16g/L of dipotassium hydrogen phosphate and 4g/L of monopotassium phosphate;
sugar is added in a flowing way: total sugar concentration 60% (W/V), total sugar concentration refers to the concentration of glucose and lactose, for example, glucose concentration 40%, lactose concentration 20%, total sugar concentration 60%; in this example, the ratio of glucose to lactose was 1:1;
pH adjusting alkaline solution: 25% ammonia, commercially available product;
pH adjusting acid solution: the preparation method comprises the steps of adopting self-made hydrolysate, and adding water to dilute the self-made hydrolysate according to the mass ratio of 1:1;
preparing a hydrolysate: corn steep liquor, bean pulp or cotton pulp are taken as raw materials, water is added until the dry matter content is 40-50%, then 98% concentrated sulfuric acid is added, the material acid ratio is 1:2-3, the materials are stirred uniformly and then react for 12-15 h at 110-120 ℃ in a reaction kettle, then the temperature is reduced to 50-55 ℃, filtration is carried out, and the filtrate is collected to obtain hydrolysate.
(2) Enzyme production by semi-continuous fermentation
The seed liquid obtained in the example 2 is inoculated into a fermentation tank filled with 40% fermentation medium according to the inoculation ratio of 0.25%, the initial fermentation temperature is 37 ℃, the pH is controlled to be 6.9-7.1 by adding pH adjusting acid-base solution, the air quantity is controlled to be 10-30L/min, the rotating speed is controlled to be 200-700 rpm, the tank pressure is controlled to be 0.05-0.1 MPa, dissolved Oxygen (DO) is controlled to be 20-30% before induction, when the bottom sugar is completely consumed, the Dissolved Oxygen (DO) is raised back, the feeding sugar is started, the fluctuation of the DO is controlled to be 30-40% by controlling the feeding sugar rate, (the sugar feeding rate of the feeding sugar is 5 g/L/h), the glucose content in the fermentation liquid is controlled to be zero, the fermentation temperature is reduced to 33 ℃, the feeding is started when the volume of the fermentation liquid reaches 50% of the volume of the fermentation tank, the 10% feeding medium is fed at the same time, the feeding culture medium is continued, the feeding sugar is continued to induce enzyme production, and the steps are repeated when the volume of the fermentation liquid reaches 50% again.
(3) Detection of cell amount and enzyme activity
And (3) taking 100mL of fermentation liquor, centrifuging at 10000rpm for 15min, collecting thalli in the fermentation liquor, and measuring the thalli quantity and the thalli enzyme activity. The test results are shown in Table 1:
TABLE 1 bacterial cell amount and bacterial cell enzyme activity detection
Example 4: semi-continuous fermentation enzyme production process
The present embodiment provides a semi-continuous fermentation enzyme production process, which is different from embodiment 3 in that: the glucose and lactose ratio in the fed-batch candy was adjusted to 2:1 (the glucose feeding rate of fed-batch candy was 3 g/L/h) and the same as in example 3.
Example 5: semi-continuous fermentation enzyme production process
The present embodiment provides a semi-continuous fermentation enzyme production process, which is different from embodiment 3 in that: the glucose and lactose ratio in the fed-batch glucose was adjusted to 3:1 (the glucose feeding rate of fed-batch glucose was 2 g/L/h) as in example 3
Comparative example 1: enzyme production by batch fermentation
The comparative example provides a method for producing enzyme by conventional batch fermentation, recombinant bacteria in the comparative example are recombinant expression strain E.coli BL21 (DE 3)/pET-28 a-treY and recombinant expression strain E.coli BL21 (DE 3) LysS/pET-24a-treZ, and the steps are as follows:
(1) Preparing materials:
fermentation medium: 25g/L of glucose, 45g/L of hydrolysate, 8g/L of dipotassium hydrogen phosphate and 2g/L of monopotassium phosphate;
inducer: lactose at a concentration of 40% (W/V);
pH adjusting alkaline solution: 25% ammonia, commercially available product;
pH adjusting acid solution: the preparation method comprises the steps of adopting self-made hydrolysate, and adding water to dilute the self-made hydrolysate according to the mass ratio of 1:1;
preparing a hydrolysate: corn steep liquor, bean pulp or cotton pulp are taken as raw materials, water is added until the dry matter content is 40-50%, then 98% concentrated sulfuric acid is added, the material acid ratio is 1:2-3, the materials are stirred uniformly and then react for 12-15 h at 110-120 ℃ in a reaction kettle, then the temperature is reduced to 50-55 ℃, filtration is carried out, and the filtrate is collected to obtain hydrolysate.
(2) Enzyme production by batch fermentation
Inoculating the seed liquid into a fermentation tank filled with 40% fermentation medium according to an inoculation ratio of 0.25%, controlling the initial fermentation temperature to 37 ℃, feeding pH-regulating acid-base solution to control pH to 6.9-7.1, controlling air quantity to 10-30L/min, controlling rotating speed to 200-700 rpm, controlling tank pressure to 0.05-0.1 MPa, controlling DO before induction to 20-30%, when the consumption of bottom sugar is complete, adding lactose to start induction after DO rises, controlling the fluctuation of air quantity, rotating speed and tank pressure to 30-40%, simultaneously reducing the fermentation temperature to 28 ℃, and inducing culture until lactose consumption completely stops fermentation.
(3) Detection of cell amount and enzyme activity
And (3) taking 100mL of fermentation liquor, centrifuging at 10000rpm for 15min, collecting thalli in the fermentation liquor, and measuring the thalli quantity and the thalli enzyme activity.
Comparative example 2: fed-batch fermentation to produce enzyme (high density fermentation)
The comparative example provides a method for producing enzyme by fed-batch fermentation, wherein recombinant bacteria are recombinant expression strains E.coli BL21 (DE 3)/pET-28 a-treY and E.coli BL21 (DE 3) LysS/pET-24a-treZ, and the steps are as follows:
(1) Preparing materials:
fermentation medium: 25g/L of glucose, 45g/L of hydrolysate, 8g/L of dipotassium hydrogen phosphate and 2g/L of monopotassium phosphate;
feeding a culture medium: 600g/L glucose, 90g/L hydrolysate, 16g/L dipotassium hydrogen phosphate and 4g/L potassium dihydrogen phosphate;
inducer: 100mM IPTG;
pH adjusting alkaline solution: 25% ammonia, commercially available product;
pH adjusting acid solution: the preparation method comprises the steps of adopting self-made hydrolysate, and adding water to dilute the self-made hydrolysate according to the mass ratio of 1:1;
preparing a hydrolysate: corn steep liquor, bean pulp or cotton pulp are taken as raw materials, water is added until the dry matter content is 40-50%, then 98% concentrated sulfuric acid is added, the material acid ratio is 1:2-3, the materials are stirred uniformly and then react for 12-15 h at 110-120 ℃ in a reaction kettle, then the temperature is reduced to 50-55 ℃, filtration is carried out, and the filtrate is collected to obtain hydrolysate.
(2) Fed-batch fermentation to produce enzymes
Inoculating the seed solution into a fermentation tank filled with 40% fermentation medium according to an inoculation ratio of 0.25%, controlling the initial fermentation temperature to 37 ℃, controlling pH to 6.9-7.1 by feeding pH-adjusting acid-base solution, controlling air quantity to 10-30L/min, controlling rotating speed to 200-700 rpm, controlling tank pressure to 0.05-0.1 MPa, controlling DO before induction to 20-30%, adding IPTG for induction when the bottom sugar consumes full DO and returns, controlling the final concentration of IPTG to 0.2mM, and controlling the specific growth rate of thalli by feeding the medium for 0.1h -1 And (3) reducing the fermentation temperature to 28 ℃, continuously adding the fermentation solution until DO is reduced to zero under the highest fermentation condition, stopping feeding, ending the fermentation, and finally enabling the volume of the fermentation liquor to reach about 60% of the volume of the fermentation tank.
(3) Detection of cell amount and enzyme activity
And (3) taking 100mL of fermentation liquor, centrifuging at 10000rpm for 15min, collecting thalli in the fermentation liquor, and measuring the thalli quantity and the thalli enzyme activity.
Under the conditions of fermentation enzyme production in different examples, the bacterial amount and bacterial enzyme activity in the fermentation broth are shown in Table 2:
TABLE 2 amount of thallus and thallus enzymatic Activity in fermentation broths of different fermentation methods
From Table 2, it can be seen that the enzyme activity of maltooligosaccharide-based trehalose synthase can be improved by 1.76-2.4 times, and the bacterial cell quantity can be improved by 1.42-1.5625 times by adopting the semi-continuous fermentation method (process) provided by the application; the enzyme activity of the mycotic maltooligosyl trehalose hydrolase is improved by 1.2-1.525 times, and the mycotic quantity is improved by 1.75-1.94 times.
Comparative example 3 different amounts of sugar blend
In the semi-continuous fermentation induction process in the comparative example, different sugar supplementing rates of mixed sugar (glucose and lactose) influence the bacterial body quantity and bacterial body enzyme activity in fermentation liquor.
This comparative example was identical to example 3 except that in this comparative example, the sugar supplement rate of fed-batch sugar during the start of fed-batch sugar was 7g/L/h, which was greater than that of example 3; or the different rate of fed-batch sugar was 2g/L/h, which is less than the rate of sugar supplementation of example 3.
When the sugar supplementing rate is smaller or larger, the bacterial amount and the bacterial enzyme activity in the fermentation liquid all show a decreasing trend, and the presumed reasons are as follows: when the sugar supplementing rate is smaller, the supplementing glucose can not meet the growth requirement of the thalli, so that the thalli grow slowly, and the supplementing inducer lactose is small in quantity, so that the induction strength is low, and the enzymatic activity of the thalli is low; when the sugar supplementing rate is high, the excessive glucose is added to cause the thalli to grow only by glucose, and lactose is not used for inducing enzyme production to cause the thalli to have low enzyme activity.
Comparative example 4 different optimization sequences
In the invention, in the example 1, a plurality of optimized sequences are obtained, and in practical experiments, 2 sequences can not successfully express the protein, or the protein expression amount in semi-continuous fermentation is similar to or even worse than the enzyme activity and yield of high-density fermentation in the prior art.
This time an optimized sequence (sequence shown as SEQ ID NO. 3) was provided to obtain the cell amount and enzyme activity in semi-continuous fermentation (method same as example 3) and batch fermentation (method same as comparative example 1) and high-density fermentation (method same as comparative example 2), as shown in Table 3.
TABLE 3 amount of thallus and thallus enzymatic Activity in fermentation broths of different fermentation methods
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A semi-continuous fermentation method is characterized in that in the semi-continuous fermentation method, mixed sugar of glucose and lactose is fed as an inducer to induce the expression of maltooligosaccharide-based trehalose synthase or maltooligosaccharide-based trehalose hydrolase in recombinant bacteria.
2. The semi-continuous fermentation process according to claim 1, wherein the total sugar concentration of the mixed sugar is 40-80%; controlling dissolved oxygen value to be 30-40% in the fed-batch mixed sugar control fermentation system; preferably, the flow acceleration of the mixed sugar is 2-5g/L/h.
3. The semi-continuous fermentation process according to claim 2, characterized in that the mass ratio of glucose to lactose is: 1:1-3:1.
4. The semi-continuous fermentation method according to any one of claims 1 to 3, wherein the recombinant bacterium contains an optimized maltooligosyl trehalose synthase encoding gene, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1; or the recombinant bacteria contains the optimized coding gene of the maltooligosyl trehalose hydrolase, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
5. The semi-continuous fermentation process according to claim 4, characterized in that it comprises:
(1) Taking escherichia coli containing the coding gene shown in any one of SEQ ID NO.1 or SEQ ID NO.2 as engineering bacteria, and inoculating the activated escherichia coli seed liquid into a fermentation medium;
(2) Starting material discharge when the volume of the fermentation liquid is 40-50% of the volume of the fermentation container, and simultaneously supplementing a fed-batch culture medium accounting for 40-60% of the material discharge amount when each material discharge is performed, and feeding sugar to induce enzyme production;
(3) And (3) repeating the step (2) when the volume of the fermentation liquor reaches 40-50% of the fermentation container again.
6. The semi-continuous fermentation process of claim 5, wherein the induction culture of step (2) comprises: before induction, the dissolved oxygen of the fermentation system is 20-30%, when the bottom sugar of the fermentation medium is completely consumed, the fed-batch sugar is started after the dissolved oxygen rises back, and the dissolved oxygen of the fermentation system is controlled to be 30-40% by the fed-batch sugar;
preferably, during the induction culture, the glucose content in the fermentation broth is zero, and the fermentation temperature is reduced by 30-33 ℃.
7. The method according to claim 6, wherein the pH is controlled to 6.9-7.1, the air volume is controlled to 10-30L/min, the rotation speed is controlled to 200-700 rpm, and the pressure in the tank is controlled to 0.05-0.1 MPa.
8. The semi-continuous fermentation process of claim 7, wherein the formulation of the fermentation medium of step (1) comprises: glucose, hydrolysate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate; the hydrolysis liquid is prepared by taking corn steep liquor, bean pulp or cotton pulp as raw materials, adding water to 40-50% of dry matter content, adding sulfuric acid, uniformly stirring, reacting for 12-15 h at 110-120 ℃ in a reaction kettle, cooling to 50-55 ℃ and filtering.
Use of the coding gene shown in any one of SEQ ID No.1 or SEQ ID No.2 or the semi-continuous fermentation method of any one of claims 1-8 for increasing the yield or enzyme activity of a recombinant maltooligosaccharide-producing trehalose synthase or maltooligosaccharide-based trehalose hydrolase.
10. Use of the semi-continuous fermentation process of any one of claims 1-8 for the synthesis of trehalose by a double enzyme process.
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