Use of lactase dt1 to degrade lactose

Abstract

The application relates to the field of agricultural biotechnology, and particularly relates to the application of lactase DT1 in degrading lactose, the application of lactose with harm into prebiotics, and the improvement of the nutritional value of milk. The lactase in the application degrades lactose in milk to generate glucose and galactose, and a transglycosylation reaction occurs to generate galacto-oligosaccharide. The content of lactose is reduced from 45 g / L to 13 g / L, and the degradation rate is more than 70%; meanwhile, more than 14 g / L of galacto-oligosaccharide with prebiotic activity is generated. Compared with common lactase, the lactase DT1 with high transglycosylation activity can not only reduce the content of lactose in milk, but also greatly reduce the content of glucose and galactose, and a large amount of galacto-oligosaccharide can be generated.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to application of lactase DT1 in degrading lactose. Background Art

[0002] Galactooligosaccharide (GOS) is an oligosaccharide composed of 2 to 10 galactoses and 1 glucose linked in sequence, which is polymerized by the transglycosidic action of lactase (also known as β-galactosidase) with lactose as the substrate.

[0003] Currently, there are over 17,000 lactase genes registered with NCBI. Enzymes from different sources have different properties. For example, lactases derived from bacteria are mostly mesophilic. For example, lactases from Escherichia coli and Lactobacillus bulgaricus generally have an optimal reaction temperature of around 40°C and an optimal pH between 6.5 and 7.5. Lactases from yeast, on the other hand, have strong hydrolytic activity and are commonly used to hydrolyze lactose in milk and whey. Furthermore, lactases from molds typically have an optimal reaction temperature above 50°C and an optimal pH slightly acidic. Lactases from Aspergillus niger are particularly heat- and acid-resistant and can be used for the hydrolysis of cheese, milk, and acid whey.

[0004] The transglycosylation activity of lactase can be used to produce galacto-oligosaccharides. However, few lactases with transglycosidic activity have been reported. A lactase with transglycosidic activity has been reported to be obtained from a strain of Aspergillus oryzae.

[0005] Although it has been reported that the prior art reports from Bifidobacterium bifidum (Bifidobacterium bifidum ) has transglycosidic properties and can hydrolyze lactose to produce galacto-oligosaccharides. However, the transglycosidic properties of lactases from different genera of Bifidobacterium vary greatly. For example, no transglycosidic activity was detected in BLGLB1 from Bifidobacterium longum; BPGLB1 from Bifidobacterium pseudocatenulatus had no transglycosidic activity; BgaC from Bifidobacterium adolescentis could undergo transglycosylation at a lower lactose concentration (~200 mM) to produce DP2-4 galacto-oligosaccharides (GOS); BIF1 from Bifidobacterium DSM20215 and INF1 from Bifidobacterium infantis DSM20088 had transglycosylation activity only at a lactose concentration of 400 mM, and the transglycosylation efficiency of BIF1 was higher than that of INF1.

[0006] Therefore, there is an urgent need to explore lactase gene resources that can hydrolyze lactose to produce oligosaccharides at low lactose concentrations and express them efficiently to reduce production costs. Summary of the Invention

[0007] The purpose of the present invention is to provide application of lactase DT1 in degrading lactose.

[0008] According to the technical solution of the present application, the amino acid sequence of the lactase DT1 is shown in SEQ ID No: 1, which is derived from Bifidobacterium leontopithec .

[0009] The reaction temperature of lactose degradation by the lactase DT1 is 45°C-50°C.

[0010] The pH at which the lactase DT1 degrades lactose is 4.0-9.0.

[0011] The lactase DT1 can be used for processing room temperature milk to produce liquid milk with low lactose and high prebiotic oligosaccharides;

[0012] Used for processing low-temperature milk fresh-keeping milk, producing warm milk fresh-keeping milk with low lactose and high beneficial oligosaccharides;

[0013] Used in milk powder processing to produce zero-lactose, high-prebiotic milk powder suitable for middle-aged and elderly people. This can solve the problem of high monosaccharide content in the past when lactase was used to prepare this type of milk powder, and can also solve the problem of high sugar content in milk; or

[0014] Lactose can also be directly used to produce prebiotics such as galacto-oligosaccharides.

[0015] >SEQ ID No: 1:

[0016] 。

[0017] The present invention uses genetic engineering to obtain a novel lactase DT1 expressed in Bacillus. This lactase has an optimal temperature of 50°C and an optimal pH of 6.0. After incubation at 45°C for 60 minutes, the lactase retains 90% activity and is relatively stable. It is inactive after incubation at 60°C for 10 minutes and at 65°C for 5 minutes. These enzymatic properties facilitate the post-processing of the enzyme in milk. This lactase can degrade lactose to produce galacto-oligosaccharides (GOS). Adding it to milk significantly reduces lactose content, generates GOS, and improves milk quality. Therefore, this lactase has broad application prospects in a variety of fields, including milk applications, food health care, medicine, and infant food. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the SDS-PAGE image;

[0019] Figure 2 Shows the optimum temperature of lactase DT1;

[0020] Figure 3 The thermostability of lactase DT1 was shown;

[0021] Figure 4 Shows the optimum pH for lactase DT1;

[0022] Figure 5 Shows the pH stability of lactase DT1;

[0023] Figure 6 The transamination characteristics of lactase DT1 under low lactose concentration conditions were demonstrated;

[0024] Figure 7 HPLC analysis showed that lactase DT1 converted 10% lactose into galacto-oligosaccharides;

[0025] Figure 8 Shows the time-dependent distribution of galacto-oligosaccharides produced by lactase DT1 using 10% lactose as substrate;

[0026] Figure 9 HPLC analysis showing the production of galacto-oligosaccharides by lactase DT1 in milk. DETAILED DESCRIPTION

[0027] The construction process of the present invention is further described below with reference to examples. The relevant examples in the following description are for illustrative purposes only and cannot limit the scope of protection of the present invention.

[0028] Example 1 Construction and expression of recombinant vector

[0029] Synthetic gene DT1 (nucleotide sequence shown in SEQ ID No: 2), with enzyme cleavage sites upstream and downstream Nde I and Eco RV sequence, and then inserted into the same restriction site of puC57. DT1 was amplified by PCR, and the recombinant vector pBCpf1 was used Pme I digestion. After recovering the digestion product and PCR product, they were ligated with a recombinase, transformed into E. coli Top10, and plated on LB plates containing 50 μg / ml kanamycin for resistance screening. PCR identification of the resulting transformants on the plates revealed positive E. coli transformants harboring the recombinant vector pBCpf1-GalA-DT1.

[0030] Lactase gene sequence:

[0031] >SEQ ID No: 2:

[0032]

[0033] Plasmid pBCpf1-GalA-DT1 was extracted from the TOP10 recombinant bacteria, methylated, and then transformed into Bacillus by electroporation. PCR analysis of the resulting transformants on the plate revealed positive Bacillus transformants harboring the recombinant vector pBCpf1-GalA-DT1.

[0034] Example 2 Fermentation and screening

[0035] 1. Plate activation of positive clones

[0036] Plate activation medium recipe: LB solid medium: tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L), and agar (20 g / L). After streaking or spreading, incubate at 30°C for 72 hours until colonies are well established.

[0037] 2. Bacteria activation

[0038] Formula of culture medium for activation of bacterial strains: LB liquid medium: tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L).

[0039] The colonies on the plates were picked and inoculated into the activated culture medium and cultured at 37°C for 48 h until the bacteria grew well.

[0040] 3. Shake flask fermentation: The culture medium formula is (g / mL): LB + 2% syrup, inoculated into the fermentation medium at a ratio of 2%, fermentation temperature controlled at 37°C, culture for 48 hours, and then determine the lactase activity.

[0041] Lactase activity was determined using the o-dianisidine method (ONPG). The strain pBCpf1-GalA-DT1-17 was screened and identified. The lactase activity in the vial was approximately 0.7 U / mL. The sample was diluted 20-fold, and the ΔOD420 value was 0.126. The SDS-PAGE image of the vial sample is shown below. Figure 1 shown.

[0042] Example 3 Lactase hydrolysis properties

[0043] 1. Determination of lactase DT1 activity: dilute the lactase solution with 0.1 mol / L pH 7.0 phosphate buffer, take 200 mL of the dilution and add 800 mL of 0.25% ONPG (o-nitrophenol-β-D-galactopyranoside), incubate in a 50°C water bath for 15 min, add 1 mL of 10% trichloroacetic acid to terminate the reaction, then add 1 mL of 1 mol / L sodium carbonate colorimetric solution, and calculate the OD 420The lactase activity was determined, and 0.1 mol / L pH 6.0 phosphate buffer was used instead of the supernatant dilution as a control. The content of p-nitrophenol in the hydrolysis product was calculated, and the enzyme activity was calculated using a standard song.

[0044] 2. Optimum temperature of lactase DT1

[0045] The optimum temperature of lactase was determined by the following method: the lactase solution was appropriately diluted with 0.1 mol / L disodium hydrogen phosphate-citrate buffer and reacted at different temperatures of 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 70°C and 80°C at pH 7.0. The thermometer at which the enzyme activity was the highest was taken as the optimum temperature.

[0046] The results are as follows Figure 2 As shown in the figure, the highest enzyme activity was taken as 100% to calculate the relative enzyme activity. It can be seen that the optimal temperature of DT1 is 50°C.

[0047] 3. Temperature stability of lactase DT1

[0048] The temperature stability of lactase was determined by the following method: the lactase solution was appropriately diluted with 0.1 mol / L sodium hydrogen phosphate-citrate buffer (pH 7.0), incubated at 37°C, 45°C, 50°C, 60°C, and 65°C for 0, 5, 10, 15, 20, 30, 45, and 60 minutes, and then further diluted with 0.1 mol / L sodium hydrogen phosphate-citrate buffer (pH 7.0) to determine the enzyme activity. The relative enzyme activity was calculated based on the enzyme activity measured at 0 minute without incubation as 100%.

[0049] The results are as follows Figure 3 As shown, lactase DT1 still has 100% relative enzyme activity after reacting at 37°C and 45°C for 1 hour, but has no enzyme activity after reacting at 65°C for 5 minutes.

[0050] 4. Optimal pH of Lactase DT1

[0051] The optimum pH of lactase was determined by the following method: the lactase solution was diluted with 0.1 mol / L different buffers with a pH range of 3.0-11.0, including 0.1 mol / L disodium hydrogen phosphate-citrate buffer for pH 3.0-8.0 and 0.1 mol / L glycine-sodium hydroxide buffer for pH 9.0-11.0. The relative enzyme activity was calculated with the highest enzyme activity being taken as 100%, and the pH value at which the enzyme activity was highest was taken as the optimum pH.

[0052] The results are as follows Figure 4 As shown, the optimum pH of lactase DT1 is 6.0.

[0053] 5. pH stability of lactase DT1

[0054] The pH stability of lactase was determined by the following method: the lactase solution was appropriately diluted with different pH 3.0-11.0 buffers (0.1 mol / L disodium hydrogen phosphate-citrate buffer for pH 3.0-8.0, 0.1 mol / L glycine-sodium hydroxide buffer for pH 9.0-11.0), incubated at 37°C for 1 hour, and then diluted with the optimal pH 6.0 buffer. The enzyme activity was measured at the optimal temperature of 50°C, and the relative enzyme activity was calculated with the highest enzyme activity being set as 100%.

[0055] The results are as follows Figure 5 As shown in Figure 2, the stable range of lactase DT1 is pH 4.0-9.0.

[0056] 6. Production of galacto-oligosaccharides at low lactose concentration

[0057] The lyophilized lactase powder was diluted 10-fold with water to an enzyme activity of 130 U / mL. The enzyme was then added to 30 mL of 125 mM, 150 mM, and 200 mM lactose solutions at concentrations of 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, and 1%, respectively. After incubation in a water bath shaker at 45°C for 1 hour, 1 mL of the reaction solution was added with 20 ul of 100% trichloroacetic acid to terminate the reaction. The final concentration of trichloroacetic acid was 2%. The solution was mixed on an oscillator and filtered through a 0.22 mm filter membrane. HPLC analysis was then performed, with lactose solutions of different concentrations not added with enzyme solution as controls.

[0058] The results are as follows Figure 6 As shown, lactase DT1 can convert lactose into galacto-oligosaccharides at low lactose concentrations of 125 mM, 150 mM, and 200 mM. The higher the lactose concentration, the higher the galacto-oligosaccharide content, reaching maximum values ​​of 1.61%, 2.03%, and 2.86%, respectively. At a relatively high lactose concentration (200 mM), the galacto-oligosaccharide content increased with increasing lactase addition and then remained constant. At lower lactose concentrations (125 mM / 150 mM), the galacto-oligosaccharide content reached its maximum at 0.5% lactase addition, and galacto-oligosaccharide production did not continue to increase with subsequent lactase addition. These results demonstrate that lactase DT1 exhibits transglycosylation properties at low lactose concentrations (125 mM / 150 mM).

[0059] 7. Comparison of properties of lactases from different bifidobacteria. The properties of lactases from different bifidobacteria are shown in Table 1 below.

[0060] Table 1 Comparison of properties of lactase from different bifidobacteria .

[0061] Among them, Document 1 is: Du M, Yang S, Jiang T, Liang T, Li Y, Cai S, et al. Cloning, Expression, Purification, and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum. Molecules.2022;27(14):4497;

[0062] Document 2 is: Mulualem DM, Agbavwe C, Ogilvie LA, Jones BV, Kilcoyne M, O'Byrne C, et al. Metagenomic identification, purification and characterization of the Bifidobacterium adolescentis BgaC β-galactosidase. AppliedMicrobiology and Biotechnology. 2021;105(3):1063–1078;

[0063] Document 3 is: Møller PL, Jørgensen F, Hansen OC, Madsen SM, Stougaard P. Intra- and Extracellular β-Galactosidases from Bifidobacterium bifidum and B.infantis: Molecular Cloning, Heterologous Expression, and ComparativeCharacterization. Applied and Environmental Microbiology. 2001;67(5):2276–2283.

[0064] BLGLB1 from B. longum, with ONPG as substrate, the optimum pH is 5.5, the optimum temperature is 45°C, the enzyme activity is 2200±15 U / mg; the relative activity is 75% after 60 minutes of treatment at 35-45°C, and the relative activity is more than 110% in the range of pH 4.0-9.0; no transglycosylation activity is detected.

[0065] The hydrolysis optimum pH of BPGLB1 from B. pseudocatenulatum is 6.0, and the optimum temperature is 45°C; the activity of BPGLB1 can be increased by more than 2 times after short-term treatment at low temperature (35-40°C), the activity of BPGLB1 decreases sharply when pH>7.0, and the residual activity is less than 10% at pH 9.0; no transglycosylation activity is detected.

[0066] The hydrolysis optimum pH of BgaC from B. adolescentis is 7.0, and the optimum temperature is 37°C; 87% of the activity is retained after 24 h of treatment at pH 4-10, 65% of the activity is retained after 1 h of treatment at 40°C, the activity is stable when stored at 4°C or -20°C, and the activity decreases by 35% when stored at room temperature for 24 h. BgaC can perform transglycosylation at a relatively low lactose concentration (~200 mM) to generate oligomeric galactose (GOS) with DP2-4.

[0067] The hydrolysis optimum pH of BIF1 from Bifidobacterium DSM20215 and INF1 from B. infantis DSM20088 is 7.0, and the optimum temperature is 37°C; both genes have transglycosylation activity at a lactose concentration of 400 mM, and the transglycosylation efficiency of BIF1 is higher than that of INF1.

[0068] The hydrolysis optimum temperature of DT1 of the present application is 50°C, and the optimum pH is 6.0; the enzyme activity remains 100% after 1 hour of incubation at 37°C and 45°C, and the relative activity is more than 80% in the range of pH 4-9. Compared with other genes from Bifidobacterium described above, lactase DT1 still has transglycosylation activity at a relatively low lactose concentration of 125 mM, which improves the economy and practicability of GOS synthesis and lays a foundation for subsequent efficient production of prebiotics.

[0069] Example 4 Degradation of lactose by lactase DT1

[0070] The lactase freeze-dried powder is diluted 10 times with water to 130 U / mL, and then added to 10% β-lactose at an amount of 0.2%, and then incubated at 30°C for 5 h, and then boiled for 10 minutes to terminate the reaction, and then centrifuged at 12000 r / min for 3 minutes, and then filtered with a 0.22 mm filter membrane, and then analyzed by HPLC.

[0071] Results as shown in Figure 7 The lactase can degrade lactose into glucose, galactose, and simultaneously synthesize oligogalactose. Among them, the contents of various oligosaccharides are as follows: oligosaccharide 54.40 g / L; galactose 2.38 g / L; lactose residual amount 29.53 g / L; glucose yield 13.69 g / L, i.e. oligosaccharide yield 54.40%, lactose degradation rate 27.38%, lactose involved in hydrolysis 38.85%; lactose involved in transglycosylation reaction 77.19%; lactase transglycosylation rate 43.09%.

[0072] The lactase freeze-dried powder was diluted 10 times with water to 130 U / mL, and then added to 10% b-lactose. After standing at 30°C for 1 h, 3 h, 5 h, 6 h, 7 h, 8 h, 15 h, 17 h, 18 h, 21 h, 26 h, and 28 h, the reaction was terminated by boiling for 10 min. Centrifugation was performed at 12000 r / min for 3 min, and the filtrate was filtered using a 0.22 mm filter membrane, followed by HPLC analysis.

[0073] Results as shown in Figure 8 It can be seen from the results as shown in that with the extension of the reaction time, the amount of oligogalactose presents a trend of first increasing and then slightly decreasing. Since lactase has both hydrolysis and transglycosylation activities, the decomposition and synthesis of GOS occur simultaneously. Within the first 7 h of the reaction, the amount of oligogalactose presents an increasing trend, and the concentration of oligogalactose can be as high as 4%. After 15 h, 10% lactose substrate can be degraded to 2.79%, and then the content of lactose presents a slight increasing trend. After 28 h of reaction, the content of lactose is 3.06%.

[0074] Example 5 Transformation of lactose in milk by lactase

[0075] 1. Transformation test of milk. The lactase freeze-dried powder was diluted 10 times with water to 130 U / mL, and then added to 30 mL of pure milk (lactose content 4.5%) in a 45°C water bath shaker. After 1 h of reaction, 1 mL of the reaction solution was added to 20 mL of 100% trichloroacetic acid to terminate the reaction. The final concentration of trichloroacetic acid was 2%, and the mixture was uniformly mixed on a shaker and centrifuged at 12000 r / min for 3 min. The filtrate was then filtered using a 0.22 mm filter membrane, followed by HPLC analysis. The milk without enzyme was used as a blank control.

[0076] 2. Detection of oligogalactose

[0077] The filtered sample was analyzed by HPLC. The conditions for HPLC analysis are as follows.

[0078] Main body: High-speed liquid chromatography (HPLC); Chromatographic column: Shodex SUGAR KS-802, 8*300mm; Mobile phase: Ultrapure water; Flow rate: 0.4 ml / min; Sample injection volume: 5 mL.

[0079] The products of lactose decomposition were analyzed by HPLC, and the relative yield of galacto-oligosaccharides and the degradation rate of lactose were calculated by converting the amounts of lactose, galactose and glucose standards.

[0080] The results are as follows Figure 9 As shown, lactase degrades lactose in milk to produce glucose and galactose, which simultaneously undergo polymerization to form galacto-oligosaccharides. The oligosaccharide contents are as follows: 14.3 g / L galacto-oligosaccharides; 4.06 g / L galactose; 13.37 g / L residual lactose; and 12.29 g / L glucose. This results in a 32.42% oligosaccharide yield, a 55.90% lactose degradation rate, 80.30% lactose involved in hydrolysis, 46.71% lactose involved in transglycosylation, and a 13.70% lactase transglycosylation rate.

[0081] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. The application of lactase DT1 in degrading lactose to generate galacto-oligosaccharides, characterized in that: The amino acid sequence of the lactase DT1 is shown in SEQ ID No:

1. The lactase DT1 is used to degrade lactose with a concentration of 125 mM to 200 mM.

2. The use according to claim 1, characterized in that The reaction temperature of the lactase DT1 for degrading lactose to generate galacto-oligosaccharides is 45°C-50°C.

3. The use according to claim 1, characterized in that The pH value at which the lactase DT1 degrades lactose to generate galacto-oligosaccharides is 4.0-9.

0.

4. The use according to claim 1, characterized in that The lactase DT1 is used to degrade lactose in room temperature milk to generate galacto-oligosaccharides.

5. The use according to claim 1, characterized in that The lactase DT1 is used to degrade lactose in low-temperature milk to generate galacto-oligosaccharides.

6. The use according to claim 1, characterized in that The lactase DT1 is used to degrade lactose in milk powder to generate galacto-oligosaccharides.

7. The use according to claim 1, characterized in that The lactase DT1 is used for preparing galacto-oligosaccharides by degrading lactose.

8. The use according to claim 1, characterized in that The lactase DT1 is prepared into a liquid or solid enzyme product.

9. The use according to claim 8, characterized in that For liquid lactase DT1, the lactase activity is 100-300 U / mL.

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