Method for preparing gentiooligosaccharide by using beta-1,6-glucanase and application of method

Abstract

The invention relates to a method for preparing gentiooligosaccharide by using beta-1,6-glucanase and an application of the method and belongs to the technical field of genetic engineering and fermentation engineering. The invention firstly provides beta-1,6-glucanase TcBgn with transglycosylation activity on gentiobiose, the beta-1,6-glucanase TcBgn can transform glucose into gentiooligosaccharide under the condition of relatively small additive amount of enzyme, and the transformation rate is high, so that the production cost can be significantly reduced. The gentiooligosaccharide is prepared by double-enzyme compounding of the beta-1,6-glucanase and beta-glucosidase, and the glucose is transformed into the gentiobiose and gentianose by using a double-enzyme compounded system, so that the transformation rate is high, and the proportion of the gentianose in a product is significantly improved. Therefore, the method for preparing the gentiooligosaccharide by a single enzyme of the beta-1,6-glucanase or double-enzyme compounding has good industrial application value.

Description

technical field

[0001] The invention relates to a method for preparing gentiooligosaccharides by using beta-1,6-glucanase and its application, and belongs to the technical fields of genetic engineering and fermentation engineering. Background technique

[0002] Gentiooligosaccharides are functional oligosaccharides formed by connecting glucose with β-1,6 glycosidic bonds, including gentiobiose and a small amount of trisaccharides and tetrasaccharides. Gentiooligosaccharides are not digested and absorbed by the human intestinal tract, but they are beneficial to the reproduction and growth of bifidobacteria and lactic acid bacteria. They are suitable for people with diabetes and other people. Their high moisturizing properties are conducive to maintaining moisture in food and preventing starch aging; At the same time, gentiooligosaccharides have high heat resistance and are suitable for foods that require high-temperature treatment; gentiotriose in its ingredients has a unique...

Examples

Embodiment 1

[0051] Example 1: Construction of β-1,6 glucanase and β-glucosidase Pichia genetically engineered bacteria

[0052] (1) Construction of β-1,6 glucanase Pichia pastoris genetically engineered bacteria

[0053] The gene encoding β-1,6-glucanase TcBgn (nucleotide sequence shown in SEQ ID NO.3) was chemically synthesized and connected to the expression vector pPIC9K of Pichia pastoris to obtain the recombinant plasmid pPIC9K-TcBgn and transform it into Escherichia coli JM109 In the middle, after enzyme digestion verification, the recombinant plasmid pPIC9K-TcBgn was integrated into Pichia pastoris KM71 by electroporation, and then the transformation solution was spread on the MD plate, and a single clone was grown on the MD plate, and then 96 transformants were picked and transferred to Transformants with higher enzyme activity were screened on a new MD plate with a small tube of 10 mL size, which was the obtained β-1,6 glucanase Pichia genetically engineered bacteria.

[0054] P...

Embodiment 2

[0059] Example 2: Fermentation production of β-glucosidase and β-1,6-glucanase in a 3.6L tank

[0060] (1) Inoculate the β-1,6 glucanase Pichia genetically engineered bacteria and the β-glucosidase Pichia pastoris genetically engineered bacteria prepared in Example 1 into YPD medium respectively, at 30°C, 200rpm Cultivate for 24 hours until the OD of the seed solution 600 It is 1.3-1.5.

[0061] (2) Batch fermentation stage: inoculate the seed solution in the fermenter with 8%-12% inoculation amount, control the temperature at 28-30°C, the initial rotation speed at 180-220rpm, the initial ventilation rate at 7L / min, and the dissolved oxygen at 28-32 %, pH 4.5-5.5;

[0062] (3) Feed-fed fermentation stage: when dissolved oxygen rises to 80-100%, feed-fed culture is carried out by adding glycerin at a constant rate, controlling temperature at 28-30°C, dissolved oxygen at 28-32%, and pH 4.5-5.5;

[0063] (4) Induction culture stage: when the bacterial cell concentration OD 60...

Embodiment 3

[0076] Example 3: Enzymatic properties of β-glucosidase

[0077] The obtained β-glucosidase enzyme liquid is characterized enzymatically by the above-mentioned enzyme activity assay method, and the enzyme activity is measured at different temperatures with pNPG as a substrate, and the results show that the optimum temperature of β-glucosidase is It is 60 DEG C; Then under optimum temperature conditions, different pH gradients are set to measure the enzymatic activity of β-glucosidase, and the optimum pH of this enzyme is 4.5( Figure 4 ).

technical field

[0001] The invention relates to a method for preparing gentiooligosaccharides by using beta-1,6-glucanase and its application, and belongs to the technical fields of genetic engineering and fermentation engineering. Background technique

[0002] Gentiooligosaccharides are functional oligosaccharides formed by connecting glucose with β-1,6 glycosidic bonds, including gentiobiose and a small amount of trisaccharides and tetrasaccharides. Gentiooligosaccharides are not digested and absorbed by the human intestinal tract, but they are beneficial to the reproduction and growth of bifidobacteria and lactic acid bacteria. They are suitable for people with diabetes and other people. Their high moisturizing properties are conducive to maintaining moisture in food and preventing starch aging; At the same time, gentiooligosaccharides have high heat resistance and are suitable for foods that require high-temperature treatment; gentiotriose in its ingredients has a unique...

Examples

Embodiment 1

[0051] Example 1: Construction of β-1,6 glucanase and β-glucosidase Pichia genetically engineered bacteria

[0052] (1) Construction of β-1,6 glucanase Pichia pastoris genetically engineered bacteria

[0053] The gene encoding β-1,6-glucanase TcBgn (nucleotide sequence shown in SEQ ID NO.3) was chemically synthesized and connected to the expression vector pPIC9K of Pichia pastoris to obtain the recombinant plasmid pPIC9K-TcBgn and transform it into Escherichia coli JM109 In the middle, after enzyme digestion verification, the recombinant plasmid pPIC9K-TcBgn was integrated into Pichia pastoris KM71 by electroporation, and then the transformation solution was spread on the MD plate, and a single clone was grown on the MD plate, and then 96 transformants were picked and transferred to Transformants with higher enzyme activity were screened on a new MD plate with a small tube of 10 mL size, which was the obtained β-1,6 glucanase Pichia genetically engineered bacteria.

[0054] P...

Embodiment 2

[0059] Example 2: Fermentation production of β-glucosidase and β-1,6-glucanase in a 3.6L tank

[0060] (1) Inoculate the β-1,6 glucanase Pichia genetically engineered bacteria and the β-glucosidase Pichia pastoris genetically engineered bacteria prepared in Example 1 into YPD medium respectively, at 30°C, 200rpm Cultivate for 24 hours until the OD of the seed solution 600 It is 1.3-1.5.

[0061] (2) Batch fermentation stage: inoculate the seed solution in the fermenter with 8%-12% inoculation amount, control the temperature at 28-30°C, the initial rotation speed at 180-220rpm, the initial ventilation rate at 7L / min, and the dissolved oxygen at 28-32 %, pH 4.5-5.5;

[0062] (3) Feed-fed fermentation stage: when dissolved oxygen rises to 80-100%, feed-fed culture is carried out by adding glycerin at a constant rate, controlling temperature at 28-30°C, dissolved oxygen at 28-32%, and pH 4.5-5.5;

[0063] (4) Induction culture stage: when the bacterial cell concentration OD 60...

Embodiment 3

[0076] Example 3: Enzymatic properties of β-glucosidase

[0077] The obtained β-glucosidase enzyme liquid is characterized enzymatically by the above-mentioned enzyme activity assay method, and the enzyme activity is measured at different temperatures with pNPG as a substrate, and the results show that the optimum temperature of β-glucosidase is It is 60 DEG C; Then under optimum temperature conditions, different pH gradients are set to measure the enzymatic activity of β-glucosidase, and the optimum pH of this enzyme is 4.5( Figure 4 ).

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