Heat-resistant lactase mutant and application thereof in synthesis of galactooligosaccharide

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a heat-resistant lactase mutant and application thereof in galactooligosaccharide synthesis. In order to overcome the defects that lactase for GOS synthesis in the prior art is insufficient in heat resistance and cannot efficiently catalyze under the conditions of high temperature and high substrate concentration, the invention provides a lactase mutant BglSS obtained through rational design, and the amino acid sequence is as shown in SEQ ID NO: 3. The mutant keeps high catalytic activity, the thermal stability is remarkably improved, GOS synthesis can be stably catalyzed for a long time under the conditions of 65-70 DEG C and 600-800 g / L high-concentration lactose, the reaction time is remarkably shortened, the production efficiency is improved, and the energy consumption and the production cost are reduced.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a thermostable lactase mutant and its application in the synthesis of galactooligosaccharides. Background Technology

[0002] Galacto-oligosaccharides (GOS) are important functional oligosaccharides widely used in infant formula and functional food industries. Industrial GOS production primarily relies on the transglycosylation reaction of lactose substrates by β-galactosidase (lactase). However, naturally derived β-galactosidases generally suffer from low expression levels and poor thermal stability, making it difficult to meet the demands of efficient and low-cost industrial production.

[0003] The applicant has conducted systematic research on the aforementioned issues and achieved several results. In Chinese invention patent application No. 202011051056.1, filed on September 29, 2020, entitled "A Special Enzyme for the Production of Galacto-Oligosaccharides and Its Preparation and Application," a highly efficient exogenous expression platform for β-galactosidase based on Bacillus licheniformis expression host and a novel vector system is disclosed, achieving high-level extracellular constitutive secretion of lactase and providing a fundamental enzyme source guarantee for industrial GOS production. In Chinese invention patent application No. 202310699268.8, filed on June 13, 2023, entitled "A Highly Active Lactase Mutant and Its Application," multiple rounds of mutation screening of β-galactosidase were conducted using molecular directed evolution technology, significantly improving the enzyme's catalytic activity and substrate adaptability, and enriching the diversity of lactase applications in GOS production. Furthermore, to achieve in-situ synthesis of GOS under low-temperature conditions such as pasteurized milk, a novel lactase mutant with high catalytic efficiency and moderate thermostability under medium- and low-temperature conditions was developed in the Chinese invention patent "A Lactase and Its Application in In-situ Conversion of Lactose in Fresh Milk" (application date: July 17, 2024, application number: 202410957962.X). This series of studies significantly broadened the preparation and application scenarios of GOS in beverage end products. While the aforementioned technologies have achieved progress in high-efficiency expression, enhanced catalytic activity, and special low-temperature applications, they have not yet provided an effective solution for the highly thermostable lactase required under high-temperature and high-substrate-concentration conditions in GOS industrial production.

[0004] The core challenge facing industrial GOS production lies in the contradiction between substrate solubility and enzyme thermal stability. The solubility of lactose in water is significantly affected by temperature, approximately 215 g / L at 20℃, approximately 320 g / L at 40℃, and approximately 640 g / L at 60℃. To increase the substrate concentration in the unit reaction system, thereby increasing product yield, reducing water evaporation in post-processing steps, and lowering production costs, industrial production typically uses a high-concentration lactose solution of 600-800 g / L as the substrate. However, high-concentration lactose solutions require temperatures of 65-70℃ or even higher to completely dissolve and maintain their dissolved state, which necessitates that the enzymatic reaction be carried out under corresponding high-temperature conditions. In addition, increasing the reaction temperature has the following process advantages: (1) According to the Arrhenius equation, the reaction rate can be increased by 1.5-2.0 times for every 10℃ increase in temperature, significantly shortening the reaction time; (2) It reduces the viscosity of the reaction system, which is beneficial for mass transfer and mixing; (3) It reduces the heating energy required to maintain a constant temperature.

[0005] However, most existing lactases are rapidly inactivated at high temperatures of 65-70°C, unable to sustain catalytic reactions for several hours. This is one of the key factors hindering the improvement of GOS industrial yield and economic benefits. Therefore, there is an urgent need to develop a heat-resistant lactase mutant with high catalytic activity, tolerance to high substrate concentrations, and long-term stability at 65-70°C to improve the industrial production efficiency and economic benefits of GOS. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing lactases used in GOS synthesis, such as insufficient heat resistance and inability to catalyze efficiently under high temperature and high substrate concentration conditions. This invention provides a heat-resistant lactase mutant, BglSS, obtained through rational design. This mutant maintains high catalytic activity while significantly improving thermal stability, enabling it to stably catalyze GOS synthesis for extended periods at 65-70℃ and a high lactose concentration of 600-800 g / L. This significantly shortens reaction time, improves production efficiency, and reduces energy consumption and production costs.

[0007] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0008] One of the technical solutions provided by the present invention is a lactase mutant, which is obtained by R27C and W49C mutations on the basis of the original lactase shown in SEQ ID NO:1; Furthermore, the lactase mutant is lactase mutant BglSS, and its amino acid sequence is shown in SEQ ID NO:3; The mutant, by introducing disulfide bonds into the protein molecule, synergistically enhances the overall structural rigidity and thermal stability of the enzyme molecule, and its heat resistance is significantly improved compared to the original enzyme.

[0009] Furthermore, the lactase mutant BglSS possesses the following enzymatic properties: (1) The optimal reaction temperature is 62℃, which is 4℃ higher than the original enzyme's 58℃; (2) The optimal reaction pH is 6.0, which is the same as the original enzyme lactase; (3) The pH stability range is 5.0-8.0. After incubation within this range for 2 hours, more than 80% of the enzyme activity is retained. (4) In a high substrate concentration (800 g / L lactose), after heat incubation at 70℃ for 1 h, the residual enzyme activity was 62.4% of the initial enzyme activity, which was 147.6% higher than the residual enzyme activity of the original lactase (25.2%). (5) The half-life at 65℃ was 45.5 min, which was 73.3% longer than the original lactase half-life of 26.2 min; (6) It can stably catalyze the synthesis of GOS for a long time under the conditions of 65-70℃ and 600-800 g / L high concentration of lactose, which significantly shortens the reaction time.

[0010] The second technical solution provided by this invention is a biomaterial, which includes any one of the following: (A1) The nucleic acid molecule of the lactase mutant as described in one of the encoding technical solutions; (A2) An expression cassette containing the nucleic acid molecule described in (A1); (A3) A recombinant vector containing the nucleic acid molecule described in (A1); (A4) Recombinant microorganisms containing the nucleic acid molecules described in (A1); Furthermore, all of the biological materials can express (A1) the nucleic acid molecules.

[0011] In the above-mentioned biological materials, the nucleic acid molecule described in (A1) includes a DNA molecule with a coding sequence as shown in SEQ ID NO:4; Furthermore, the nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID NO:4; The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as mRNA or hnRNA.

[0012] In the aforementioned biological materials, the recombinant vector described in (A3) can be constructed using an expression vector. The structure of expression vectors is well known to those skilled in the art. Expression vectors typically contain elements required for target gene expression, such as promoters, multiple cloning sites, terminators, and ribosome binding sites, and may also contain selection marker genes (such as kanamycin resistance genes). kanr Neomycin resistance gene neo Hygromycin resistance gene hyg Chloramphenicol resistance gene cat Streptomycin resistance gene str Bleomycin resistance gene ble (etc.). The expression vector can be constructed using any method known in the art (such as recombination technology, synthesis technology, etc.) or can be commercially purchased. For example, in one or more embodiments of the present invention, the recombinant vector uses the expression vector pHY-WZX.

[0013] The recombinant vector can be a recombinant expression vector obtained by cloning the nucleic acid molecule encoding the lactase mutant BglSS into an expression vector (such as a prokaryotic expression vector or a eukaryotic expression vector). Although the expression vector used in the embodiments provided by the present invention is the pHY-WZX vector, the present invention is not limited to this specific vector. Those skilled in the art can use other suitable vectors (e.g., pUB110, pE194, pHT1469, pWH1520, pHY300plk, etc.), as long as the vector can express the lactase mutant. Preferably, the present invention provides a recombinant expression vector pUBglSS, which is obtained by expressing (A1) the nucleic acid molecule on the expression vector pHY-WZX; In the above-mentioned biological materials, the recombinant microorganisms mentioned in (A4) include hosts such as Bacillus licheniformis, yeast, or Escherichia coli; Preferably, in the above-mentioned biological materials, the recombinant microorganism in (A4) can be a recombinant Bacillus licheniformis; the recombinant Bacillus licheniformis is a lactase mutant BglSS with the amino acid sequence shown in SEQ ID NO:3 expressed in a Bacillus licheniformis host; preferably, the present invention provides a recombinant Bacillus licheniformis BliBglSS, which is a strain obtained by transforming the recombinant expression vector pUBglSS into Bacillus licheniformis BCBT0529.

[0014] The third technical solution provided by this invention is the application of the lactase mutant described in the first technical solution, or the nucleic acid molecule, expression cassette, recombinant vector, or recombinant microorganism described in the second technical solution, in any of the following: (B1) Application in increasing the yield of microbial galactooligosaccharides; (B2) Application in the preparation of galactooligosaccharides or products containing galactooligosaccharides; (B3) Application in the construction of genetically engineered bacteria that produce galactooligosaccharides; Especially its application in the catalytic synthesis of galactooligosaccharides under high temperature and high substrate concentration conditions; further, the high temperature condition is 65-70℃, and the high substrate concentration condition is lactose concentration not exceeding 800 g / L, preferably 600-800 g / L.

[0015] The fourth technical solution provided by the present invention is the application of the recombinant vector or recombinant microorganism described in the second technical solution in the lactase mutant described in the first technical solution of the production technology; Furthermore, the application includes the process of culturing the recombinant microorganism in a culture medium and collecting the lactase mutant from the culture; Furthermore, the method for preparing the lactase mutant using the recombinant microorganism is as follows: (1) Fermentation expression: The recombinant microorganism was inoculated into a fermentation medium and fermented to obtain the lactase mutant; Furthermore, the shake-flask fermentation culture conditions were: 37-42℃, 200-600 r / min shaking culture for 72-120 h; Further, the fermentation medium is composed of: 3-5% (w / v) peptone, 2-3% (w / v) phosphate, 1-2% (w / v) citric acid, 0.5-2% (w / v) ammonium sulfate, 2-10% (w / v) glucose, and pH 7.0-7.2; Furthermore, glucose is added during fermentation to maintain the glucose concentration in the system at 4-6%; Furthermore, the fermentation conditions in the fermenter are as follows: temperature 37-42℃, glucose supplementation during fermentation while maintaining a sugar concentration of not less than 1%, ammonia water to control pH not less than 6.8, and dissolved oxygen level not less than 20%; The expression system is an extracellular constitutive secretory expression system, in which the target enzyme is automatically secreted into the culture medium during the culture process; (2) Fermentation broth treatment: Centrifuge the fermentation broth from step (1) and take the supernatant, which is the crude enzyme solution containing the lactase mutant. Further, the fermentation broth was centrifuged at 4000-10000 r / min for 10-15 min, and the supernatant was collected; Because an extracellular constitutive secretory expression system is used, the crude enzyme solution has extremely low levels of impurities and high enzyme purity. (3) Promoting disulfide bond formation: The crude enzyme solution obtained in step (2) is treated with hydrogen peroxide (H2O2) solution with a concentration of 0.1-1.0 mM at 4-25℃ for 30-60 min to promote the complete formation of disulfide bonds; (4) Enzyme preparation: The enzyme solution after step (3) is concentrated, freeze-dried or a protective agent is added to prepare an enzyme preparation, which can be directly used for industrial production of GOS.

[0016] Beneficial effects Compared with the original lactase BcBG168-D, the lactase mutant BglSS provided by this invention has the following advantages: Significantly improved heat resistance: The lactase mutant BglSS, by introducing disulfide bonds, has a half-life of 45.5 min required to lose half of its enzyme activity at 65℃, which is 73.7% longer than that of the original lactase BcBG168-D, and its thermal stability is significantly improved.

[0017] Thermal stability in industrial applications: After 1 h at 800 g / L lactose and 70℃, the residual enzyme activity of the lactase mutant BglSS was 62.4%, which is about 1.5 times higher than that of BcBG168-D. The lactase mutant BglSS is suitable for industrial production conditions with high temperature and high substrate concentration.

[0018] Catalytic activity retention: The specific enzyme activity of the lactase mutant BglSS is 96% of that of BcBG168-D, avoiding the activity loss caused by improving thermal stability.

[0019] Significantly improved production efficiency: At higher substrate concentrations (600-800 g / L lactose solution) and higher reaction temperatures (65-70℃, enzyme dosage 5-10 U / g substrate), the reaction time of the lactase mutant BglSS is shortened to 5-10 h, which is 30-50% shorter than that of BcBG168-D, and the highest GOS conversion rate reaches 57.8%. It is suitable for the industrial-scale, efficient production of GOS, significantly reducing production costs and energy consumption, resulting in significant economic benefits. Attached Figure Description

[0020] Figure 1 The molecular structure and disulfide bond characteristics of the lactase mutant BglSS Wherein, A: the interaction between the substrate lactose and the enzyme molecule; B: the mutated site in the enzyme molecule structure; C: the structural characteristics of disulfide bond formation after mutation.

[0021] Figure 2 SDS-PAGE images of the lactase mutant BglSS and the original lactase BcBG168-D Wherein, M: protein molecular weight standard; 1: original lactase BcBG168-D; 2: lactase mutant BglSS.

[0022] Figure 3 The thermostability curves of the lactase mutant BglSS and the original lactase BcBG168-D are shown.

[0023] Figure 4 Effects of temperature and pH on enzyme activity Where a: the effect of temperature on enzyme activity; b: the effect of pH on enzyme activity; c: the pH stability of the enzyme.

[0024] Figure 5 The reaction curves show the process of lactose synthesis into galactooligosaccharides catalyzed by the lactase mutant BglSS and the original lactase BcBG168-D under high temperature conditions. Where, a: reaction process curve at 65℃; b: reaction process curve at 70℃. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] 1. The technical route for achieving the objective of this invention is summarized as follows: Based on the original lactase BcBG168-D, this invention uses computer-aided analysis and design to obtain possible disulfide bond formation sites, and further expresses it in Bacillus licheniformis. The enzymatic properties of the obtained lactase mutant are then tested, and finally a lactase mutant BglSS with significantly improved heat resistance and catalytic activity at high substrate concentrations is obtained.

[0027] 2. Nomenclature of amino acids and DNA nucleic acid sequences The IUPAC nomenclature, a widely accepted system for naming amino acid residues, is used, employing three-letter / single-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0028] 3. Principles for Identifying (Nomenclature) Lactase Mutants The mutated amino acid in the lactase mutant is represented by "original amino acid residue + residue position + replaced amino acid residue". For example, R27C indicates that the amino acid at position 27 is replaced by cysteine ​​(C) in the original lactase BcBG168-D, and the position number corresponds to the amino acid sequence number of the original lactase BcBG168-D in SEQ ID NO: 1.

[0029] In this invention, lowercase italics bcbg168-d Indicates the gene encoding the primitive lactase BcBG168-D, in lowercase italics. bglss The encoding gene for the lactase mutant BglSS is shown in Table 1 below.

[0030] Table 1

[0031] In this invention, the original lactase BcBG168-D has been disclosed in Chinese invention patent application number 202011051056.1, filed on September 29, 2020, entitled "An enzyme for the production of galactooligosaccharides and its preparation and application," with the amino acid sequence shown in SEQ ID NO: 1. MKSTTSAAGKSVSYNDGERRVNFENWRFQRETNGSIAGAQNPGFDDSSWRKLNLPHEWSIDLDFNKNSLATHEGGYLDGGIGWNRKTFTIPESMKGKRFSLD FDGVYMNSTTYLNGEVLGTYPFGYNAFSYDISDKLYKDGRANVLVVKVNTNQPSGRWYSGSSIYRNVYLTTVDPIHVARYGTFVTTPNLEKSIKEDRDAVNIK TKISNAEAKQVKIAIKSTIYDGAGNTVQTVETEEKTAAAGTVTPFEQNTVIKQPKLWSIDKPYRYNLVTEVIVGGQTVDTYETKFGVRYFKFDENEGYSLNG EFMKLHGVSMHHDLAGGAALTNARGVERQMQIMKDVGVNAIRVTHNPASPELGLEFAANKLLIIEEAFDSWAQSKKPYDYGRFFNAWAEHDIKEMVDRGKNEP AIIMWSIGNEIYDTTNAAGVETARNLVGWVKEIDTTPRATIGEDKTRGDNVTPINSYIKEIFNIVDVVGLNYSENNYDGYHKQNPSWKLYGSETSSATRSRG VYTHPYQYNHDTKYADLQQSSYDNDYVGWGRTAQDAWKYDRDLKHIAGQFIWTGFDYIGEPTPYYNSYPAKSSYFGAVDTAGFPKDIFYYYQSQWKKEPMVHL LPHWNWKEGEKVRVLAYTNASKVELVLNGESLGNEKYDNKQTSWGAPYKETKDGKTYLEWAVPFKPGKLEAVAKDENGKVIARDQVVTAGEPASVRLTADRK VVKADGTDLSFITADIVDSKGIVVPDADHLITFNVTGQGELAGVDNGNASSVERYKDNKRKAFSGKALAIVQSSKLSGKITVHASVAGLSSDSTSVFTVTPAD In this invention, the lactase mutant BglSS has the amino acid sequence shown in SEQ ID NO: 3: MKSTTSAAGKSVSYNDGERRVNFENWCFQRETNGSIAGAQNPGFDDSSCRKLNLPHEWSIDLDFNKNSLATHEGGYLDGGIGWNRKTFTIPESMKGKRFSLDFDGVYMNSTTYLNGEVLGTYPFGYNAFSYDISDKLYKDGRANVLVVKVNTNQPSGRWYSGSSIYRNVYLTVTDPIHVARYGTFVTTPNLEKSIKEDRDAVNIKTKISNAEAKQVKIAIKSTIYDGAGNTVQTVETEEKTAAAGTVTPFEQNTVIKQPKLWSIDKPYRYNLVTEVIVGGQTVDTYETKFGVRYFKFDENEGYSLNGEFMKLHGVSMHHDLAGGAALTNARGVERQMQIMKDVGVNAIRVTHNPASPELGLEFAANKLLIIEEAFDSWAQSKKPYDYGRFFNAWAEHDIKEMVDRGKNEPAIIMWSIGNEIYDTTNAAGVETARNLVGWVKEIDTTPRATIGEDKTRGDNVTPINSYIKEIFNIVDVVGLNYSENNYDGYHKQNPSWKLYGSETSSATRSRGVYTHPYQYNHDTKYADLQQSSYDNDYVGWGRTAQDAWKYDRDLKHIAGQFIWTGFDYIGEPTPYYNSYPAKSSYFGAVDTAGFPKDIFYYYQSQWKKEPMVHLLPHWNWKEGEKVRVLAYTNASKVELVLNGESLGNEKYDNKQTSWGAPYKETKDGKTYLEWAVPFKPGKLEAVAKDENGKVIARDQVVTAGEPASVRLTADRKVVKADGTDLSFITADIVDSKGIVVPDADHLITFNVTGQGELAGVDNGNASSVERYKDNKRKAFSGKALAIVQSSKLSGKITVHASVAGLSSDSTSVFTVTPAD. 4. Sources of plasmids and strains The lactase mutant of the present invention is obtained by cloning the coding gene of the lactase mutant into the expression vector pHY-WZX (Niu DD, Wang ZX. Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis In J Ind Microbiol Biotechnol (2007) 34:357-362.DOI 10.1007 / s10295-0204-x, a recombinant lactase-producing bacterium was obtained by genetically transforming Bacillus licheniformis strain BCBT0529 (published in Chinese invention patent "A Special Enzyme for the Production of Galacto-oligosaccharides and Its Preparation and Application" with application date September 29, 2020, application number 202011051056.1). The enzyme solution was prepared by shake-flask fermentation and purified. The relevant enzymatic properties and biochemical characteristics were compared and analyzed, and its application in the preparation of GOS was studied and analyzed.

[0032] 5. The method and definition for determining lactase activity used in this invention. Lactase activity was determined according to the national standard GB / T 33409-2016. The reaction was carried out at pH 5.0 and 40℃, using o-nitrophenyl-β-D-galactose as the substrate. The amount of o-nitrophenol produced was determined by ultraviolet spectrophotometry.

[0033] Lactase activity is defined as the amount of enzyme that catalyzes the conversion of one micromolar o-nitrobenzene-β-D-galactose per minute at pH 5.0 and 40°C, and is defined as one enzyme activity unit (U), expressed as U / mL or U / g.

[0034] Unless otherwise stated, the materials, reagents and methods used in the embodiments of this invention are conventional in the art.

[0035] The present invention will be further explained and illustrated below through specific embodiments.

[0036] Example 1: Construction of lactase mutant BglSS and its recombinant strain Based on computer-aided simulation analysis, it was determined that the original lactase BcBG168-D structure lacks native disulfide bonds. Further computer analysis and disulfide bond design software (Disulfide by Design 2.0 or Yosshi) were used to obtain possible disulfide bond formation sites. Homology modeling of simulated mutations was performed on selected sites, and simulated molecular docking analysis was conducted on the protein structure. Finally, the lactase mutant BglSS (shown in SEQ ID NO: 3) was selected. Figure 1 After mutation, the interaction between the enzyme molecule and its substrate lactose molecule remains unchanged, and disulfide bonds can be formed within the molecule.

[0037] Further, based on the amino acid sequence shown in SEQ ID NO: 1, codon optimization and chemical synthesis were performed to obtain the encoding gene of the full-length lactase mutant BglSS. bglss That is, the nucleic acid molecule shown in SEQ ID NO:4.

[0038] The gene encoding the lactase mutant BglSS bglss The correct recombinant plasmid pUBglSS was obtained by restriction enzyme digestion and cloning into the expression vector pHY-WZX, followed by transformation of the intermediate host Bacillus subtilis WB600.

[0039] The correct recombinant plasmid pUBglSS was transformed into Bacillus licheniformis BCBT0529, plated on LB solid medium containing kanamycin (50 μg / mL) and X-gal (80 μg / mL), and cultured at 37℃ for 48 h. Blue colonies were screened to obtain the recombinant expression strain, which was named recombinant BliBglSS.

[0040] Using the same method described above, the encoding gene of the original lactase BcBG168-D was... bcbg168-d The recombinant plasmid was obtained by restriction enzyme digestion and cloning into the expression vector pHY-WZX, and then transformed into the intermediate host Bacillus subtilis WB600. The correct recombinant plasmid was then transformed into Bacillus licheniformis BCBT0529 to obtain the recombinant expression strain, which was named recombinant strain BliBg168-D.

[0041] Example 2: Fermentation expression and enzyme preparation of lactase mutant BglSS 1. Fermentation for lactase production: Single colonies of the recombinant bacteria BliBglSS and BcBG168-D obtained in Example 1 were inoculated into 5 mL of LB medium and cultured at 37℃ and 220 r / min for 12 h as seed for subsequent fermentation.

[0042] The seed culture was inoculated at a rate of 2% into 50 mL of fermentation medium and cultured at 37°C with shaking at 220 r / min for 72 h. The fermentation medium consisted of 4% (w / v) peptone, 2.5% (w / v) phosphate, 1.5% (w / v) citric acid, 1% (w / v) ammonium sulfate, and 5% (w / v) glucose, with a pH of 7.0 and no antibiotics added.

[0043] 2. Fermentation broth enzyme activity assay: After fermentation, the fermentation broth was centrifuged at 8000 r / min for 10 min, and the supernatant was collected as lactase enzyme solution (crude enzyme solution). The enzyme activity of the original lactase BcBG168-D and the lactase mutant BglSS was measured using the aforementioned method. The results showed that the enzyme activity of BcBG168-D in the supernatant was 39 U / mL, and the enzyme activity of the lactase mutant BglSS was 36 U / mL.

[0044] 3. Disulfide bond formation promotion: Hydrogen peroxide was added to the BglSS lactase mutant enzyme solution obtained above to a final concentration of 0.5 mM, and treated at 4℃ for 60 min to promote complete disulfide bond formation. Then, residual H2O2 was removed by ultrafiltration (10 kDa molecular weight cutoff).

[0045] 4. Concentrate the lactase mutant BglSS enzyme solution and the control BcBG168-D enzyme solution after step 3 to an enzyme activity of 100-200 U / mL, add a protective agent (10% glycerol) to prepare a liquid enzyme preparation, and store at 4℃; freeze-dry the enzyme solution directly to prepare solid enzyme powder, and store at room temperature.

[0046] Example 3: Fermentation Validation in a 30 L Fermenter 1. A 30 L fermenter is used, containing fermentation medium, with a volume of 20 L of medium per 30 L fermenter. The medium is sterilized at 121℃ for 30 min. The fermentation medium formula includes: glucose 20 g / L, yeast extract 15 g / L, peptone 10 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1.5 g / L, trace element solution 1 mL / L, pH 7.0. The trace element solution composition includes: FeSO4·7H2O 5.0 g / L, MnSO4·H2O 1.6 g / L, ZnSO4·7H2O 1.4 g / L, CoCl2·6H2O 0.4 g / L. 2. Fermentation process Inoculation: The recombinant strain BliBglSS seed culture prepared in Example 2 was aseptically inoculated into the fermenter at a volume percentage of 5%. Fermentation conditions: temperature 42℃, aeration rate 1.0 vvm, stirring speed 200-600 r / min, initial pH 7.0. During fermentation, glucose was supplemented to maintain a sugar concentration of no less than 1%, ammonia was used to control the pH to no less than 6.8, and dissolved oxygen level to no less than 20%. After 96 h of fermentation, the highest enzyme activity reached 663 U / mL.

[0047] Example 4: Enzymatic properties of lactase mutant BglSS The liquid enzyme preparation prepared in step 4 of Example 2 above was further purified using an AkTa chromatography system to obtain purified lactase mutant BglSS and original lactase BcBG168-D, which appeared as a single band on gel electrophoresis. Figure 2 The purified lactase was used in subsequent experiments.

[0048] 4.1 Determination of the thermal stability of the lactase mutant BglSS The purified lactase mutant BglSS and the original lactase BcBG168-D were diluted in 50 mM phosphate buffer (pH 6.5, enzyme concentration 0.1 mg / mL) and incubated at 65℃ for 2 h. Residual enzyme activity was measured periodically using the aforementioned enzyme activity assay method (relative residual enzyme activity was calculated with each original enzyme activity as 100%). The results are as follows: Figure 3 As shown, their half-lives are summarized in Table 2.

[0049] Table 2. Comparison of the thermal stability of the lactase mutant BglSS in the experimental environment.

[0050] It can be seen that the time required for the lactase mutant BglSS to lose half of its enzyme activity at 65℃ (half-life) is 73.7% longer than that of the original lactase BcBG168-D, indicating a significant improvement in thermal stability. Therefore, the introduction of disulfide bonds in this invention significantly enhances the thermal stability of the enzyme molecule.

[0051] 4.2 Thermal stability characteristics of lactase mutant BglSS and original lactase BcBG168-D under simulated industrial production conditions To evaluate the thermostability of the mutants in industrial applications at higher temperatures, the lactase mutant BglSS and the original lactase BcBG168-D (8 U / g substrate) were added to a reaction system of 800 g / L lactose (pH 6.5) and incubated at 70 °C for 1 h. Residual enzyme activity was then measured according to the aforementioned enzyme activity assay method, and the results are summarized in Table 3. The results showed that after 1 h at 800 g / L lactose and 70 °C, the lactase mutant BglSS retained 62.4% of its enzyme activity, approximately 1.5 times higher than that of BcBG168-D (Table 3).

[0052] Table 3. Comparison of thermal stability of lactase mutant BglSS under simulated industrial production conditions.

[0053] 4.3 Optimal Temperature and Optimal pH The optimal reaction conditions for the mutant and the original enzyme were determined by measuring enzyme activity under different temperature (50-75℃) and pH (4.0-8.0) conditions. The results are summarized in […]. Figure 4 .

[0054] The optimal operating temperature of the lactase mutant BglSS is slightly increased, by 4℃ compared to the original lactase BcBG168-D. Figure 4 (a)

[0055] The optimal pH for the lactase mutant BglSS is 6.0, and the optimal pH for the original lactase BcBG168-D is also 6.0. Figure 4 (b) The pH stability range is 5.0-8.0. After incubation within this range for 2 hours, more than 80% of the enzyme activity is retained. Figure 4 (c). It can be seen that this mutation has no significant effect on the pH properties of the enzyme.

[0056] 4.4 Enzyme activity assay The specific enzyme activities were measured at their respective optimal reaction temperatures and pH conditions for the original enzyme BcBG168-D and the lactase mutant BglSS, and the results are shown in Table 4. The introduction of disulfide bonds had a slight impact on the enzyme's catalytic activity, but still maintained 95.5% of the specific activity of BcBG168-D, which is within an acceptable range. This indicates that the disulfide bond introduction site was chosen appropriately and did not significantly interfere with the function of the catalytic center.

[0057] Table 4. Changes in specific enzyme activity of lactase mutant BglSS

[0058] 4.5 Dynamic Parameters The initial reaction rates of the original lactase BcBG168-D and the lactase mutant BglSS were determined using different concentrations of lactose substrate (10-500 mmol / L). Kinetic parameters were calculated using the Lineweaver-Burk double reciprocal plot method, and the results are summarized in Table 5. The kinetic parameters of all lactase mutants, BglSS, were essentially equivalent to those of the original enzyme BcBG168-D, indicating that the introduction of disulfide bonds to improve thermal stability did not significantly affect the enzyme's binding affinity to the substrate or its catalytic conversion efficiency.

[0059] Table 5. Changes in enzyme kinetic parameters of the lactase mutant BglSS

[0060] Example 5: Application of the lactase mutant BglSS primitive enzyme BcBG168-D in the synthesis of GOS at high temperature and high substrate concentration. GOS synthesis was carried out at 65°C, 600 g / L lactose, and 6 U / g substrate (the enzyme preparation prepared in step 4 of Example 2). Samples were taken periodically, and the GOS content was analyzed by HPLC. The GOS yield was calculated, and the results are as follows: Figure 5 As shown in Figure a.

[0061] The results showed that the lactase mutant BglSS reached a peak GOS conversion rate of 57.8% at 6 h, while the original lactase BcBG168-D reached 49.5% at 12 h under the same conditions. Figure 5(a) The lactase mutant BglSS shortens the reaction time by 50% while maintaining a high conversion rate.

[0062] GOS synthesis was carried out at 70°C, 800 g / L lactose, and 8 U / g enzyme substrate (the enzyme preparation prepared in step 4 of Example 2). Samples were taken periodically, and the GOS content was analyzed by HPLC. The GOS yield was calculated, and the results are as follows: Figure 5 As shown in b.

[0063] The results showed that the lactase mutant BglSS achieved a GOS conversion rate of 57.2% at 8 h, while the original lactase BcBG168-D experienced a rapid decline in enzyme activity under the same conditions, with a maximum conversion rate of only 28.3%, demonstrating the significant advantage of the lactase mutant BglSS under extreme reaction conditions.

[0064] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A lactase mutant, characterized in that, The lactase mutant is lactase mutant BglSS, which is obtained by R27C and W49C mutations on the original lactase shown in SEQ ID NO:

1.

2. A biomaterial, characterized in that, The biomaterial includes any one of the following (A1)-(A4): (A1) A nucleic acid molecule encoding the lactase mutant of claim 1; (A2) An expression cassette containing the nucleic acid molecule described in (A1); (A3) A recombinant vector containing the nucleic acid molecule described in (A1); (A4) A recombinant microorganism containing the nucleic acid molecule described in (A1), wherein the recombinant microorganism uses Bacillus licheniformis BCBT0529 as a host.

3. A biomaterial as described in claim 2, characterized in that, (A1) The nucleic acid molecule includes a DNA molecule with a coding sequence as shown in SEQ ID NO:4; The lactase mutant BglSS has the amino acid sequence shown in SEQ ID NO:

3.

4. A biomaterial as described in claim 2, characterized in that, (A3) The expression vectors used in the recombinant vector include: pHY-WZX, pUB110, pE194, pHT1469, pWH1520 or pHY300plk.

5. A biomaterial as described in claim 2, characterized in that, (A3) The recombinant vector is the recombinant expression vector pUBglSS obtained by expressing the nucleic acid molecule described in (A1) on the expression vector pHY-WZX; (A4) The recombinant microorganism is a strain obtained by transforming the recombinant expression vector pUBglSS into Bacillus licheniformis BCBT0529.

6. The use of the lactase mutant of claim 1, or the nucleic acid molecule, expression cassette, recombinant vector, or recombinant microorganism of claim 2, in any one of the following (B1)-(B3): (B1) Application in increasing the yield of microbial galactooligosaccharides; (B2) Application in the preparation of galactooligosaccharides or products containing galactooligosaccharides; (B3) Application in the construction of genetically engineered bacteria that produce galactooligosaccharides.

7. The application as described in claim 6, characterized in that, It is used in the catalytic synthesis of galactooligosaccharides under high temperature conditions of 65-70℃ and / or in the catalytic synthesis of galactooligosaccharides under lactose concentration conditions not exceeding 800 g / L.

8. The use of the recombinant vector or recombinant microorganism of claim 2 in the production of the lactase mutant of claim 1.

9. The application as described in claim 8, characterized in that, The method for preparing the lactase mutant using the recombinant microorganism is as follows: (1) Fermentation expression: The recombinant microorganisms were inoculated into a fermentation medium and fermented to obtain a fermentation broth; (2) Fermentation broth treatment: Centrifuge the fermentation broth obtained in step (1) and take the supernatant, which is the crude enzyme solution containing the lactase mutant; (3) Disulfide bond formation promotion: The crude enzyme solution obtained in step (2) is treated with hydrogen peroxide solution with a concentration of 0.1-1.0 mM at 4-25℃ for 30-60 min to promote the complete formation of disulfide bonds and obtain the lactase mutant.

10. The application as described in claim 9, characterized in that, The enzyme solution after step (3) is concentrated, freeze-dried, or a protective agent is added to prepare an enzyme preparation, which can be directly used for the industrial production of GOS.

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