WO2022042005A1 - Souche recombinante produisant une amylase maltogénique et son application dans des aliments cuits - Google Patents

Souche recombinante produisant une amylase maltogénique et son application dans des aliments cuits Download PDF

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WO2022042005A1
WO2022042005A1 PCT/CN2021/103084 CN2021103084W WO2022042005A1 WO 2022042005 A1 WO2022042005 A1 WO 2022042005A1 CN 2021103084 W CN2021103084 W CN 2021103084W WO 2022042005 A1 WO2022042005 A1 WO 2022042005A1
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maltogenic amylase
recombinant
bread
mutant
blma
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Rongzhen ZHANG
Yan Xu
Yingqi RUAN
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/80—Pastry not otherwise provided for elsewhere, e.g. cakes, biscuits or cookies
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01133—Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/10—Bacillus licheniformis

Definitions

  • the present disclosure relates to recombinant strains producing maltogenic amylase and its application in baked foods, belonging to the technical fields of enzyme engineering and food engineering.
  • Maltogenic amylase (EC 3.2.1.133) , belonging to glycoside hydrolase family 13, is a type of endo-acting amylase that hydrolyzes the ⁇ -1, 4-glucosidic bond.
  • Maltogenic amylase enzymes can hydrolyze various substrates such as cyclodextrins, starches and pullulan to maltose, exhibiting multi-substrate specificity and catalytic versatility.
  • Maltogenic amylase can be isolated from various sources, including Bacillus licheniformis, Bacillus subtilis, Bacillus stearothermophilus, Bacillus cereus, Thermus sp., and Thermus vulgaris, etc. Different sources of maltogenic amylase have great differences in enzymatic properties.
  • maltogenic amylase can hydrolyze starch to maltose, oligosaccharide and small molecule dextrin. These maltose and small molecule dextrin are too short to crystallize and form crystalline junction zones, interfere with the recrystallization of starch and the entanglement of starch granules and protein macromolecules, thereby reducing the regenerating rate and recrystallization rate of starch granules, keeping the bread soft and its freshness longer. At present, maltogenic amylase is gradually being used in the bakery industry, but high-quality and safe maltogenic amylase is rarely reported.
  • B. subtilis has strong protein exocrine ability and has no obvious codon preference. It has been recognized by the US Food and Drug Administration and China's food safety related departments as a food safety-grade strain GRAS (Generally recognized as safe) . Therefore, it is a prefect expression strain for food-grade. But there exist problems such as low expression level and low recombinase activity by B. subtilis expressing maltogenic amylase. Therefore, the screening and construction of the recombinant maltogenic amylase in B. subtilis with high protein production, enzyme activity, excellent enzymatic properties are of great significance for the baked food.
  • the present disclosure screened a strain of B. licheniformis RZ108 that produces maltogenic amylase, used chromosome walking technology to clone the complete maltogenic amylase gene from its genomic DNA, and constructed an expression vector.
  • the target gene was expressed in B. subtilis WB600, and a recombinant maltogenic amylase with high expression and high enzyme activity was obtained.
  • the recombinant maltogenic amylase was applied to bread baking, the results showed it can improve the rheological properties of the dough, effectively delay the regeneration of starch, reduce the hardness of the bread during storage, and significantly improve the quality of the bread, thereby increasing the shelf life of the baking.
  • the present disclosure provides a maltogenic amylase with the amino acid sequence set forth in SEQ ID NO. 1.
  • the present disclosure provides a gene for coding the maltogenic amylase, and the nucleotide sequence set forth in SEQ ID NO. 2.
  • the present disclosure provides a recombinant plasmid carrying the gene of maltogenic amylase.
  • the vector included but was not limited to pMA0911.
  • the present disclosure provides a host cell carrying the gene of maltogenic amylase.
  • the present disclosure provides a host cell carrying the recombinant plasmid.
  • the present disclosure provides a recombinant microbial cells expressing the maltogenic amylase.
  • the recombinant microorganism was a recombinant B. subtilis, taking pMA0911 as a vector to express the maltogenic amylase in B. subtilis WB600.
  • the present disclosure provides a recombinant B. subtilis, expressing the maltogenic amylase, and the recombinant B. subtilis takes pMA0911 as an expression vector.
  • the present disclosure provides a method for constructing the recombinant bacteria, which included the following steps: (1) ligating the maltogenic amylase gene to the vector pMA0911, (2) transforming the obtained recombinant expression vector into B. subtilis WB600 to obtain recombinant bacteria.
  • the complete maltogenic amylase gene was obtained by using chromosome walking technology.
  • restriction enzymes EcoR I and BamH I were used to digest the target gene and plasmid.
  • lysozyme and a plasmid extraction kit were used to extract recombinant plasmids and to verify positive clones of recombinant bacteria.
  • B. subtilis was transformed by an improved Spizizen method.
  • the present disclosure provides a method for soluble expression of maltogenic amylase, which inoculated the recombinant bacteria into a culture medium and cultured it at 30-35°C for 24 to 48 h.
  • culturing the recombinant bacteria at 33 °C for 48 h culturing the recombinant bacteria at 33 °C for 48 h.
  • the medium contains tryptone, yeast extract, glycerol, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.
  • the medium formula was 12 g/L tryptone, 24 g/L yeast extract, 5 g/L glycerol, 2.3 g/L potassium dihydrogen phosphate and 12.5 g/L potassium dihydrogen phosphate, pH 7.0.
  • an york protein purification system was used for protein purification and DNS method was used to determine the enzyme activity.
  • the present disclosure provides an enzyme preparation, wherein it contains the pure maltogenic amylase or a mixture of the maltogenic amylase and an enzyme protective agent.
  • the present disclosure provides an application of the maltogenic amylase, or the recombinant B. subtilis, or the enzyme preparation, or recombinant microbial cells in the field of bakery products.
  • the baked products included but was not limited to baked bread.
  • the dough formula was 500 g flour, 35 g white sugar, 5 g edible salt, 5 g yeast, and 650 g water.
  • the bread baking formula was as follows: 1,000 g flour, 70 g white sugar, 10 g edible salt, 10 g yeast, and 650 g water.
  • the present disclosure provides a directed evolution method, which obtains a maltogenic amylase with better thermo-stability.
  • error-prone PCR technology was used to amplify the target gene and construct a mutant gene library, two rounds of screening were used to obtained ideal mutants, and the gene sequence of the obtained mutant was performed.
  • gene single-site-directed mutagenesis and multi-site-directed mutagenesis technology were used to obtain target mutants.
  • the ideal mutants were obtained by comparing the enzyme activity and optimal temperature of all mutants
  • the present disclosure provides a maltogenic amylase mutant, comprising an amino acid sequence with mutations of the 418 th lysine and the 296 th valine, wherein the mutations are relative to a parent amino acid sequence set forth in SEQ ID NO. 1.
  • the maltogenic amylase mutant comprising an amino acid sequence with mutations of the 418 th lysine and the 296 th valine, wherein the mutations are relative to a parent amino acid sequence set forth in SEQ ID NO. 1. and the 296 th position is mutated from valine to phenylalanine, the 418 th position is mutated from lysine to isoleucine, the amino acid sequence of the maltogenic amylase mutant is set forth in SEQ ID NO. 9.
  • the present disclosure provides a gene for coding the above-mentioned maltogenic amylase mutant.
  • the present disclosure provides a recombinant plasmid carrying the above-mentioned gene.
  • the present disclosure provides a recombinant microbial cell expressing the above-mentioned maltogenic amylase mutant.
  • the present disclosure provides a recombinant B. subtilis, expressing the maltogenic amylase mutant, and the recombinant B. subtilis takes pMA0911 as an expression vector.
  • the present disclosure provides an application of the above-mentioned mutant or the above-mentioned recombinant B. subtilis in the field of bakery products.
  • the bakery products include but is not limited to bread.
  • the present disclosure constructed the recombinant strain B. subtilis WB600/pMA0911-BLMA, which could efficiently express the target gene, and applied it to baked food.
  • the crude enzyme solution expressed by recombinant bacteria was purified by ammonium sulfate gradient sedimentation and His-Trap HP chromatography column to obtain pure recombinant enzyme BLMA.
  • the optimum temperature of pure enzyme BLMA is 60 °C. In the temperature range of 30-80 °C, more than 60%of the enzyme activity is remained after 30 min incubation.
  • the optimum pH is 6.5, over 60%of the enzyme activity can be retained in the range of pH 5.5-7.5.
  • the activity of pure enzyme BLMA is as high as 3235.0 U/mg.
  • the maltogenic amylase BLMA produced by the recombinant bacteria has the characteristics of high enzyme activity, good thermo-stability and a wide range of pH applications.
  • the enzyme activity of the most ideal mutant was increased to 2.16 times, and the optimum temperature was increased to 65 °C.
  • Fig. 1 The hardness of bread during storage.
  • Fig. 2 The elasticity of bread during storage.
  • Fig. 3 The activity and optimal temperature of enzyme for single-site-directedmutagenesis.
  • Fig. 4 Enzyme activity of double site-directed mutants.
  • Fig. 5 Optimum temperature of maltogenic amylase produced by strains 5-8.
  • Fig. 7 The hardness and elasticity of bread during storage.
  • Fig. 8 The sensory score of bread.
  • Example 1 Isolation of maltogenic amylase-producing strain
  • Liquid medium 5 g/L NaCl, 0.1 g/L MgSO 4 , 0.5 g/L KH 2 PO 4 , 0.2 g/L CaCl 2 , 0.3 g/L yeast extract, 0.3 g/L tryptone, pH 6.0.
  • Plate medium 0.2%soluble starch, 1%protein, 0.5%NaCl, 0.3%beef extract, 2% agar, pH 7.0.
  • Lugol's solution 0.3%I 2 , 0.6%KI.
  • Isolation method Soil samples with rich starch were cultured in liquid medium and incubated at 37 °C, with 200 rpm shaking for 24 h. 1 mL of culture solution was taken and diluted with physiological saline (1 ⁇ 10 -1 , 1 ⁇ 10 -2 , 1 ⁇ 10 -3 , 1 ⁇ 10 -4 , 1 ⁇ 10 -5 ) , the diluent was spread on the screening plate, and cultured at 30 °C for 56 h. Lugol's solution was added to the plate at 20 °C, and the clones with the prevalent hydrolysis circle were selected. The first screened strains were inoculated in LB liquid medium, and cultivated at 30 °C, 200 rpm for 12 h.
  • 3%of the inoculum was inoculated at 50 mL enzyme production medium, cultivated at 30 °C, 200 rpm for 48 h, and placed in a refrigerated centrifuge at 8,000 rpm, centrifuged for 5 min, and then took the fermentation supernatant to determine the enzyme activity.
  • the isolate was identified using bacteriological tests.
  • the morphological, physiological and chemical properties of the strain were analyzed, and the strain was initially identified as Bacillus sp.
  • the strain was determined to be B. licheniformis, named B. licheniformis RZ108.
  • the protein produced by B. licheniformis RZ108 was analyzed by SDS-PAGE, and the protein band produced by maltogenic amylase was cut and digested with trypsin. The released short peptide sequence was analyzed by MALDI-TOF-MS to obtain the partial amino acid sequence of maltogenic amylase.
  • Primer synthesis P1: AARWSNAARTGGAARATGT (SEQ ID NO. 3) ; P2: NTTYACNGTNAARWSNGCNC (SEQ ID NO. 4) ; P3: TTTGACGGCGTCGATGC (SEQ ID NO. 5) ; P4: GCGAATGGTATGGCG (SEQ ID NO. 6) ; P5: TTCCAGATGGTTGGCCGT (SEQ ID NO. 7) ; P6: TGAGCGTCAACAGCAACA (SEQ ID NO. 8) .
  • PCR conditions 98 °C thermal denaturation 30 s; 98 °C 15 s, 55 °C 15 s, 72 °C 2 min; after 30 cycles, 72 °C extends for another 5 min.
  • the degenerate primers P1 and P2 were designed to amplify part of the sequence of the gene with B. licheniformis RZ108 genomic DNA as template and P1 and P2 as primers.
  • two rounds of inverse PCR were applied to characterize the flanking sequences of the known fragments.
  • the genomic DNA of B. licheniformis RZ108 was digested with Sma I, following by the self-ligation under conditions conducive to form the monomer loops by T4 DNA ligase.
  • the first round of inverse PCR was performed with primers P3 and P4 to amplify the flanking fragment (0.96 kb) .
  • the second round of inverse PCR was performed using the RZ108 genomic DNA digested by Nco I as template, with primers P5 and P6, to amplify the flanking fragment (1.1 kb) .
  • the possible start codon ATG is located in the 0.96 kb fragment
  • the possible stop codon TAA is located in the 1.1 kb fragment
  • the catalytic triad Asp-Glu-Asp is located in the 1.1 kb fragment. Assemble the three fragments to obtain the complete coding gene BLMA (1,707 bp) .
  • the plasmid pMA0911 was extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing BioDev-Tech. Co., Ltd) .
  • the water, buffer, plasmid pMA0911and enzyme were added by order to the Eppendorf tube, and the tube was shaken to mix the liquid thoroughly.
  • the tube was placed in a centrifuge for 2 s to concentrate the liquid on the bottom of the tube, incubated at 37 °C for 1 h. Then the tube was kept at 65 °C for 10 min to terminate the digestion reaction.
  • the digested products were analyzed by agarose gel electrophoresis, and the gel was recovered and concentrated.
  • Enzyme digestion system 30 ⁇ L plasmid, 13 ⁇ L sterile water, 5 ⁇ L 10 ⁇ quick-cut enzyme buffer, 1 ⁇ L restriction endonuclease EcoR I, 1 ⁇ L restriction endonuclease BamH I.
  • the reaction system of connection between the target gene and the plasmid T-BLMA and PMA0911 0.5 ⁇ L T4 DNA Ligase, 1 ⁇ L 10 ⁇ T4 DNA Ligase buffer, plasmid and gene in a molar ratio of 1: 7, total 8 ⁇ L. Put the mixed connection solution in a 16 °C incubator and connect for 30 min.
  • TB medium 12 g/L tryptone, 24 g/L yeast extract, 5 g/L glycerol, 2.3 g/L potassium dihydrogen phosphate, 12.5 g/L dipotassium hydrogen phosphate, 50 ⁇ g/mL kanamycin sulfate, pH 7.0.
  • the positive clone B. subtilis WB600/pMA0911-BLMA was picked and then placed in 20 mL TB liquid medium and cultured at 33°C for 48 h.
  • the cultured recombinant B. subtilis cells were centrifuged at 4 °C, with 8,000 rpm shaking for 15 min to remove the bacteria, and the fermentation supernatant was collected.
  • Ammonium sulfate of 30% was slowly added to the supernatant. After 4 h at 4 °C, the supernatant was centrifuged with 8,000 ⁇ g shaking at 4 °C for 10 min to remove the contaminated proteins. A portion of ammonium sulfate powder was added to the supernatant to make the final concentration of ammonium sulfate in the supernatant 50%. After standing at 4 °C for 6 h, the supernatant was centrifuged at 10,000 ⁇ g at 4 °C for 30 min to collect the precipitate.
  • the precipitate was reconstituted with 20 mM Tris-HCl buffer (pH 8.0) , and then further purified using protein purification system, and the solution was loaded onto a HisTrap HP column (GE Healthcare, Chicago, USA) .
  • the column was eluted with 20 mM phosphate buffer (pH 8.0) containing 20 mM imidazole and 500 mM NaCl, and the collected target protein solution (about 2 mL) was concentrated with an ultrafiltration tube (Millipore Amicon Ultra, Billerica, USA) .
  • the imidazole was removed from the column by using desalting, and the protein purity was judged by SDS-PAGE analysis.
  • Maltogenic amylase can hydrolyze starch into reducing sugars, and reducing sugars can form brown-red amino complexes with 3, 5-dinitrosalicylic acid under heating conditions. Within a certain range, the amount of reducing sugars and the color of the reaction solution is directly proportional.
  • a spectrophotometer can be used for colorimetric measurement at 540 nm to obtain the sugar content in the sample.
  • the enzyme was incubated in phosphate buffer (pH 6.5) under a temperature gradient of 40 to 90 °C.
  • the enzyme activity of maltogenic amylase BLMA at different temperatures was calculated and summarized in Table 1.
  • the highest enzyme activity is 3235.0 U/mg, in the range of 55-75 °C, the enzyme activity can still retain more than 75%of the total activity.
  • the optimum pH of BLMA was determined in buffers with different pH: 50 mM citrate buffer (pH 4.0-6.0) , 50 mM phosphate buffer (pH 6.0-7.0) and 50 mM Tris-HCl buffer (pH 7.0-9.0) , the measurement temperature is 60 °C.
  • the enzyme activity of BLMA measured at different pH was shown in Table 2. In the pH range of 5.0-8.0, the enzyme activity of the recombinase reaches more than 80%of the total activity, indicating that BLMA has a wider pH adaptation range.
  • pH Enzyme activity (U/mg) 4.0 672.4 4.5 1684.7 5.0 2630.0 5.5 2818.4 6.0 3078.8
  • the temperature of the bread can reach above 95 °C, while after BLMA is treated at 90 °C for 30 min, the residual enzyme activity is 0, which means BLMA will not cause the baked product to sticky.
  • BLMA's wide application temperature range and good temperature stability provide huge potential advantages for its application in the bread baking industry.
  • pH Remaining enzyme activity (%) 4.0 12 4.5 20 5.0 58 5.5 69 6.0 75 6.0 80 6.5 100 7.0 86 7.0 79 7.5 68 8.0 29 8.5 20 9.0 14
  • Example 14 Determination of rheological properties of dough
  • Dough formula 500 g flour, 35 g white sugar, 5 g edible salt, 5 g yeast, and 650 g water.
  • the temperature control includes three phases: (1) constant temperature phase: 30 °C for 8 min; (2) heating phase: within 15 min, the temperature is raised to 90 °C at 4 °C/min and maintained at high temperature for 7 min; (3) cooling stage: the temperature is reduced to 50 °C at a rate of 4 °C/min within 10 min, and the temperature is maintained for 5 min.
  • maltogenic amylase BLMA 3235.0 U/mg
  • the effects of maltogenic amylase BLMA (3235.0 U/mg) on the dough quality were investigated by adding different amounts of enzyme (0, 30, 60, and 90 ppm) to the dough.
  • the sample without treatment of enzyme was used as control.
  • the water absorption rate of the dough was measured first, according to the water absorption rate, an appropriate amount of maltogenic amylase was added for the experiment, and finally C1, C2, C3, C4 and C5, as well as the formation time, stability time and other parameters were shown in Table 6.
  • C1 maximum dough consistency
  • C2 protein weakening under mechanical forces and temperature degree
  • T1 the dough formation time
  • Ts the stability time that the torque produced is kept at 1.1 Nm
  • C1-C2 total weakening value
  • C3 the gelatinization of starch during heating
  • C4 the stability of starch gelatinized adhesive
  • C5 the starch retrogradation during the cooling stage
  • C3-C4 starch breakdown
  • C5-C4 starch retrogradation at cooling stage.
  • the amount of recombinant BLMA had a slight impact on the formation time.
  • the stability time Ts of the dough has increased, because the longer the stability time of the dough, the better the toughness, quality and applicability of the dough, therefore, adding 60 ppm of BLMA is beneficial to provide dough strength and mixing endurance.
  • the peak viscosity of the dough C3 didn’t change much, but there were significant differences in the viscosity disintegration value C3-C4 and the retrogradation value C5-C4 (p ⁇ 0.05) .
  • the C5-C4 value was lower, the retrogradation of starch was lower.
  • the dough made using 60 ppm BLMA exhibited the lowest value of C5-C4 (1.26) , adding the recombinant enzyme at 60 ppm effectively reduces the retrogradation of starch.
  • Bread formula 1,000 g flour, 70 g white sugar, 10 g edible salt, 10 g yeast, and 650 g water.
  • Baking bread process First, all the ingredients were put into the dough mixer (Hauswirt, Qingdao, China) . The dough was kneaded at a low speed for 4 min, and at a high speed for 2 min. Put the dough into the SM-40SP proofer (Xinmai Machinery Co. Ltd China) for 40 min (30 °C, 85%RH, first proof) . After the dough was relaxed for 5 min, it was divided into pieces and proofed for 30 min (30 °C, 85%RH, second proof) . The final dough temperature was about 26-27 °C. Next, the dough from the container was removed, and was covered with a thin, clean plastic, and kept for about 5 min.
  • the bread was baked in a Z202 oven (Demax Technology Co., Ltd. Foshan, China) at temperature of 170 °C.
  • the baking time was 32 min. After freshly baked bread was cooled for 1 h, it was placed in a polyethylene bag and placed at 25 °C and 33%RH.
  • the specific volume of bread reflects the proofing and holding power of the dough: the larger the specific volume of bread, the easier the bread is to proof. It was measured according to the method described in AACC, 10-05 (AACC, 2000) . Specific volume (SV) was calculated as the ratio bread apparent volume (V) and mass (m) . The specific volume of the four groups of bread was shown in Table 7. The specific volumes of bread in groups A (60 ppm BLMA) , B (90 ppm MAM100) and C (60 ppm Novamyl 3D BG) were significantly higher (p ⁇ 0.05) than those of the control group D by 32.0%, 24.7%, and 30.3%, respectively.
  • the maltogenic amylase hydrolyzed the starch to maltose during the dough fermentation.
  • the maltose was then hydrolyzed to produce glucose by maltase from the baker's yeast, thus accelerating the proliferation of the yeast, and increasing gas production thus improving the specific volume of the bread.
  • the hardness of bread was evaluated by a texture analyzer TA-XT 2 (Shanghai BosinTech Co. Ltd) according to AACC74-09 (AACC, 2000) . After the breads were stored for 0, 3, 5, 7 and 10 days, they were cut into 12.5 mm thick slices from the center. The two slices were stacked together as a sample for the test of their hardness and elasticity. Each 2 cm thick slice was fixed on the stage and compressed to 50%of the height using a cylindrical aluminum probe with a diameter of 25 mm.
  • the hardness of bread was calculated from force-distance diagrams.
  • the effect of BLMA on bread hardness was shown in Figure 1. Compared with the control group D, the bread hardness of groups A, B and C with added maltogenic amylase was significantly reduced by 2.12, 1.52 and 1.98 times, respectively, after 10 days of storage. Group A with the recombinant maltogenic amylase BLMA of the present disclosure had a better hardness during storage.
  • Directed evolution method Error-prone PCR technology was used to amplify the target gene.
  • DNA polymerase to amplify the target gene, by artificially adjusting the reaction conditions, such as increasing the concentration of Mn 2+ , changing the concentration of the four dNTPs in the system, or using low-fidelity DNA polymerase to change the amplification, so as to randomly introduce mutations into the target gene at a certain frequency to obtain random mutants of protein molecules.
  • PCR conditions 94 °C thermal denaturation 10 s; 98 °C 10 s, 55 °C 5 s, 72 °C 2 min; after 30 cycles, 72 °C extends for another 5 min.
  • R TTTAAGGATCCTTACTGGATCGTTTTGCCGGCCG (SEQ ID NO. 11) .
  • the gene expression vector was constructed to obtain the gene mutation library, and the mutation library was screened by measuring the enzyme activity of the crude enzyme solution. By comparing the enzyme activity of mutants and BLMA, the mutants with higher enzyme activity were obtained in the preliminary screening; the optimum temperature of the preliminary screening mutants was measured, and the mutants with higher optimum temperature were obtained in the second screening. Through screening, two strains with significantly improved enzyme activity or optimum temperature were obtained, named F1 and F2, respectively, as shown in Table 11. Taking the enzyme activity of BLMA before mutation as 100%, its optimum temperature is 60 °C. After the mutation, the optimum temperature of the F1 mutant was increased to 65 °C, the enzyme activity of F2 has greatly increased to 1.84 times.
  • the mutants F1 and F2 were sequenced and identified. The sequencing results of F1 and F2 were compared and analyzed with the original gene of BLMA, a total of 8 gene mutation sites were discovered, as shown in Table 12.
  • Mutants with one mutation site Under the premise of known mutation sites, PCR-mediated site-directed mutagenesis technology was used to obtain eight mutants with only one mutation site, the mutants are numbered 1-8 according to the amino acid sequence. Using BLMA-pMA0911 as a template, single-point mutations were performed using the whole plasmid PCR method. The primer design is shown in Table 13 (the mutation site is shown as the underline) .
  • PCR conditions 98 °C thermal denaturation 30 s; 98 °C 15 s, 55 °C 15 s, 72 °C 2 min; after 30 cycles, 72 °C extends for another 5 min.
  • Reaction system 25 ⁇ L PrimeSTAR, 22 ⁇ L ddH 2 O, 1 ⁇ L Primers F, 1 ⁇ L Primers R, 1 ⁇ L DNA.
  • the qualified mutants were screened by measuring the enzyme activity and optimum temperature of the mutants. Taking the enzyme activity of BLMA as 100%, the enzyme activity and optimum temperature of mutants were shown in Figure 3. The enzyme activity was not improved better than F2 and the optimum temperature was not increased higher than F1.
  • Mutants with two mutation sites The random combination method was used to determine the mutation site combination, and the multi-site-directed mutagenesis technology was used to obtain mutants with two mutation sites.
  • the mutants that meet requirements were screened by measuring the enzyme activity and temperature of the mutants. Taking the enzyme activity of BLMA as 100%, the enzyme activity was shown in Figure 4, The enzyme activity of strains 5-8 was increased to 2.16 times that of BLMA. The optimum temperature and thermo-stability of the mutant strain 5-8 were shown in Figure 5 and Figure 6. The optimum temperature was higher than that of BLMA, and retained more than 75%of the total activity in the range of 55 °C to 80 °C.
  • the maltogenic amylase produced by this mutant strain 5-8 was named BLMA1, the BLMA1 maltogenic amylase mutant, comprising an amino acid sequence with mutations of the 418 th lysine and the 296 th valine, wherein the mutations are relative to a parent amino acid sequence set forth in SEQ ID NO.
  • the amino acid sequence of the maltogenic amylase mutant BLMA1 is set forth in SEQ ID NO. 9.
  • BLMA1 had more sufficient hydrolysis and produces more effective small molecule products, which reduced the recrystallization rate of starch.
  • the moisture contained in the bread also makes the bread more elastic.
  • the sensory scores of the bread on the 7th day of storage were shown in Figure 8.
  • the mutant BLMA1 improved the appearance, color, texture, smell and taste of bread, BLMA1 was more conducive to bread moisturizing, the skin color and appearance of the bread would be brighter and more attractive when stored for 7 days, which was more likely to arouse the appetite of the tester for bread. In terms of taste, the better elasticity of bread made the bread taste better.
  • the bread with the 45 ppm BLMA1 maintained better quality during storage, and it would be more economical to apply the mutant BLMA1 to bread baking at a lower concentration.

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Abstract

L'invention concerne une souche recombinante produisant de l'amylase maltogénique et son application dans des aliments cuits, appartenant aux domaines techniques de l'ingénierie génétique, de l'ingénierie enzymatique et de l'ingénierie alimentaire. L'activité enzymatique de l'amylase maltogénique recombinante BLMA a un taux de 3 235,0 U/mg à 60°C et un pH de 6,5 avec une bonne stabilité de pH et une large plage de température. Grâce à l'utilisation d'une PCR sujette aux erreurs, des technologies de mutagenèse dirigée par des gènes, un mutant présentant une meilleure activité enzymatique et une meilleure température optimale ont été obtenus, l'activité enzymatique du mutant BLMA1 a été augmentée de 2,16 fois et la température optimale a été augmentée à 65°C. L'application de BLMA et de BLMA1 à des aliments cuits permet à la fois de retarder efficacement le vieillissement des aliments cuits et d'améliorer la durée de conservation des aliments.
PCT/CN2021/103084 2020-08-27 2021-06-29 Souche recombinante produisant une amylase maltogénique et son application dans des aliments cuits Ceased WO2022042005A1 (fr)

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CN109982576A (zh) * 2016-09-23 2019-07-05 杜邦营养生物科学有限公司 低PH活性α-1,4/1,6-糖苷水解酶作为反刍动物的饲料添加剂用于增强淀粉消化的用途
WO2019193102A1 (fr) * 2018-04-05 2019-10-10 Dsm Ip Assets B.V. Alpha-amylase maltogénique variante
CN111132553A (zh) * 2017-08-29 2020-05-08 诺维信公司 表达抗老化/保鲜淀粉酶的面包酵母
CN111148841A (zh) * 2017-08-30 2020-05-12 诺维信公司 M35家族的内切蛋白酶和s53家族的外切蛋白酶在淀粉发酵中的组合使用
CN111944785A (zh) * 2020-08-27 2020-11-17 江南大学 一种产麦芽糖淀粉酶的重组菌株及其在烘焙食品中的应用

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CN108102996B (zh) * 2018-02-12 2020-11-06 江南大学 一种在枯草芽孢杆菌中高效表达麦芽糖淀粉酶的方法

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CN109982576A (zh) * 2016-09-23 2019-07-05 杜邦营养生物科学有限公司 低PH活性α-1,4/1,6-糖苷水解酶作为反刍动物的饲料添加剂用于增强淀粉消化的用途
CN111132553A (zh) * 2017-08-29 2020-05-08 诺维信公司 表达抗老化/保鲜淀粉酶的面包酵母
CN111148841A (zh) * 2017-08-30 2020-05-12 诺维信公司 M35家族的内切蛋白酶和s53家族的外切蛋白酶在淀粉发酵中的组合使用
WO2019193102A1 (fr) * 2018-04-05 2019-10-10 Dsm Ip Assets B.V. Alpha-amylase maltogénique variante
CN111944785A (zh) * 2020-08-27 2020-11-17 江南大学 一种产麦芽糖淀粉酶的重组菌株及其在烘焙食品中的应用

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