Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Special enzyme for galactooligosaccharide production as well as preparation and application thereof

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: 2021-03-30
森大(天津)生物科技有限公司
View PDF3 Cites 6 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Special enzyme for galactooligosaccharide production as well as preparation and application thereof
  • Special enzyme for galactooligosaccharide production as well as preparation and application thereof
  • Special enzyme for galactooligosaccharide production as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0069] 10. Preparation method of lactase enzyme preparation

[0070] After the fermentation is finished, the bacterial cells are removed by plate and frame filtration, and then the enzyme liquid is obtained after filtration by an ultrafiltration system.

[0071] 11. Determination of lactase activity

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

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

[0074] 12. Synthesis and product analysis of galactooligosaccharides

[0075] Use 300g / L-800g / L lactose as the substrate, add 5U / g-20U / g lactase, carry out the reaction at 50°C-70°C, and take sa...

Embodiment 1

[0085] Example 1: Molecular evolution of lactase

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

[0087] By truncating the coding genes of BglD305, BglD and BcBG168 to different degrees, and efficiently expressing the modified sequence and the original sequence, the corresponding gene sequence was amplified by PCR amplification technology, and cloned into the expression vector pHY-WZX, Obtaining 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 -8, pHY-Bgl-9, pHY-Bgl-10, pHY-Bgl-11, pHY-Bgl-12. The above-mentioned recombinant plasmids were trans...

Embodiment 2

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

[0093] Deletion of the aprE gene in Bacillus licheniformis CCTCC NO: M208236. With Bacillus licheniformis CCTCC NO: M208236 genomic DNA as a template, using apr-up1 (sequence 30) and apr-up2 (sequence 31) and primers apr-dn1 (sequence 32) and apr-dn2 (sequence 33) as primers, respectively The upper and lower homology arm fragments were amplified, and the sizes were 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 using the DNA of the gel recovery product as a template to obtain a deletion mutant cassette △aprE with a size of ~1.2kb. After the mutant cassette was purified and digested with Xba I, it was cloned into plasmid pT2 tsThe Sma I and Xba I sites were transformed into Escherichia coli JM109 competent cells, cultured on LB plates containing 20 μg / mL kanamycin, and the correct deletion plasmid pT2...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
degree of polymerizationaaaaaaaaaa
Login to View More

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to lactase for generating galactooligosaccharide as well as preparation and an application of the lactase.According to the invention, lactase (BglD305 derived from bacillus circulans B2301 and BglD derived from bacillus circulans ATCC 31382) molecules from two sources are taken as the basis for molecularevolution, so as to obtain lactase new enzyme molecules with high galactooligosaccharide synthesis efficiency and good expression performance; lactase is constructed, the lactase high-producing strainis constructed, the lactase can be efficiently synthesized during the submerged fermentation, the zymoprotein molecule is secreted into the culture medium, the high-activity enzyme preparation is directly prepared from the fermentation supernatant, and the lactase expression level can achieve 2208 U / mL; the fermentation manufacturing cost of lactase is reduced, the fermentation manufacturing process is simplified, and the quality of the lactase preparation is improved.

Description

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

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com