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A special enzyme for galacto-oligosaccharide production and its preparation and application

A lactase and recombinant strain technology, applied in the field of enzyme engineering, can solve the problems of difficulty in separation and purification, increase the difficulty of preparing lactase enzyme preparations, and difficult to realize the secretion and expression of lactase, and achieve the effect of reducing the cost of fermentation and manufacturing

Active Publication Date: 2022-07-19
森大(天津)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example: when expressed in Escherichia coli, the expression level of lactase is 1~3U / mL, it is difficult for this recombinant bacteria to secrete the synthesized lactase into the fermentation broth, which increases the difficulty of preparation of lactase enzyme preparation
Expressed in Pichia pastoris (Pichia pastoris) GS115, the expression level of shake flask fermentation can reach 70U / mL, but it is also difficult to realize the secretion and expression of lactase, and the separation and purification of the enzyme is extremely difficult

Method used

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  • A special enzyme for galacto-oligosaccharide production and its preparation and application
  • A special enzyme for galacto-oligosaccharide production and its preparation and application
  • A special enzyme for galacto-oligosaccharide production and its preparation and application

Examples

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Effect test

preparation example Construction

[0069] 10. Preparation method of lactase enzyme preparation

[0070] After the fermentation, the bacteria were removed by plate and frame filtration, and then the enzyme liquid was obtained by filtration through an ultrafiltration system.

[0071] 11. Lactase enzyme activity assay

[0072] The enzyme activity assay of lactase was improved according to the national standard GB / T 33409-2016. As a general procedure, the reaction was carried out at pH 5.0 and 40°C with lactose as substrate. Glucose release was measured using a biosensor.

[0073] The enzymatic activity of lactase is defined as the amount of enzyme required to decompose lactose to generate 1 micromol of glucose per minute at pH 5.0 and 40 °C, defined as one unit of enzyme activity (U), expressed in U / mL or U / g.

[0074] 12. Synthesis and product analysis of galactooligosaccharides

[0075] Using 300g / L~800g / L lactose as the substrate, adding 5U / g~20U / g lactase, the reaction is carried out at 50℃-70℃, and sampli...

Embodiment 1

[0085] Example 1: Molecular evolution of lactase

[0086] Using the genes encoding BglD305 and BglD shown in SEQ ID NO.1 and SEQ ID NO.7 of the sequence table as templates, DNAshuffling method was used to carry out molecular evolution. After enzyme activity screening, a lactase enzyme molecule BcBG168 (nucleotide sequence SEQ ID NO. 13) with significantly increased enzyme activity level was obtained, and its amino acid sequence (amino acid sequence SEQ ID NO. 14).

[0087] By truncating the coding genes of BglD305, BglD and BcBG168 to different degrees, and expressing the modified sequences and original sequences efficiently, the corresponding gene sequences were amplified by PCR amplification technology, and cloned into the expression vector pHY-WZX, Lactase expression plasmids pHY-Bgl-1, pHY-Bgl-2, pHY-Bgl-3, pHY-Bgl-4, pHY-Bgl-5, pHY-Bgl-6, pHY-Bgl-7, pHY-Bgl were obtained -8, pHY-Bgl-9, pHY-Bgl-10, pHY-Bgl-11, pHY-Bgl-12. The above recombinant plasmids were respectively ...

Embodiment 2

[0092] Example 2: Genetic modification of expression host cells

[0093] Deletion of the aprE gene in Bacillus licheniformis CCTCC NO: M208236. Bacillus licheniformis CCTCC NO: M208236 genomic DNA was used as the template, apr-up1 (sequence 30) and apr-up2 (sequence 31) and primers apr-dn1 (sequence 32) and apr-dn2 (sequence 33) were used as primers, respectively. The upper and lower homology arm fragments were amplified with sizes of 667bp and 495bp, respectively. After the PCR products of the correct size were obtained, they were purified by gel recovery, and overlapping PCR was performed with the DNA of the gel recovered product as a template to obtain a deletion mutation cassette ΔaprE with a size of ∼1.2 kb. The mutant cassette was purified and digested with Xba I, then cloned into plasmid pT2 tsThe Sma I and Xba I sites were transformed into E. coli JM109 competent cells and cultured on LB plates containing 20 μg / mL kanamycin to obtain the correct deletion plasmid pT2-...

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Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a lactase for generating galacto-oligosaccharides and its preparation and application. The present invention carries out molecular evolution on the basis of two sources of lactase (BglD305 derived from Bacillus circulans B2301 and BglD derived from Bacillus circulans ATCC 31382) molecules to obtain high efficiency of synthesizing galacto-oligosaccharides and good expression performance and construct a high-yielding strain of lactase, which can efficiently synthesize lactase and secrete the enzyme protein molecule into the medium during submerged fermentation, and directly prepare a highly active enzyme preparation from the fermentation supernatant. The expression level can reach 2208U / mL, which is helpful to reduce the fermentation manufacturing cost of lactase, simplify the fermentation manufacturing process and improve the quality of the lactase enzyme preparation.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a lactase for generating galacto-oligosaccharides and its preparation and application. Background technique: [0002] Oligosaccharides, also known as oligosaccharides, refer to linear or branched carbohydrates with a degree of polymerization of 2-10 linked by monosaccharide molecules through glycosidic bonds. They can be simply divided into functional oligosaccharides and ordinary oligosaccharides. category. Among them, functional oligosaccharides (Functional oligosaccharides) refers to the polymerization of 2-10 identical or different monosaccharides with glycosidic bonds; they have the sweet taste and sensory properties common to sugars, and can directly replace sucrose as dessert ingredients. However, it is not degraded by human gastric acid and gastric enzymes, and is not absorbed in the small intestine, but can reach the large intestine; oligosacchar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/75C12N1/21C12P19/00C12P19/04C12R1/10C12R1/07
CPCC12N9/2402C12Y302/01108C12N15/75C12P19/00C12P19/04C12P21/02C12P19/12C12N9/2471C12Y302/01023C12R2001/09C12R2001/10C12N1/205Y02P60/87
Inventor 王正祥牛丹丹田康明
Owner 森大(天津)生物科技有限公司
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