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

Method for biosynthesizing xylose

A biosynthesis and xylose technology, applied in the field of bioengineering, can solve problems such as low conversion rate, and achieve the effects of high conversion rate, simple production process and mild reaction conditions

Pending Publication Date: 2021-11-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to enzyme activity and specificity problems, the conversion rate of some rare sugars is still very low

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
  • Method for biosynthesizing xylose
  • Method for biosynthesizing xylose
  • Method for biosynthesizing xylose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0039] Embodiment 1BFD, GALS, FSA gene acquisition, vector construction

[0040] The source of the BFD (benzoylformate decarboxylase) gene is Pseudomonas putida, and its amino acid is shown in SEQID NO.1. Under the premise of not changing the amino acid sequence of BFD, the codon of the above wild-type gene is replaced with the E. coli preference (High-frequency use) codons, after codon optimization, the gene sequence has Escherichia coli preferred codons, and its gene sequence is shown in SEQ ID NO.2. The gene sequence was directly synthesized on the pET-28a vector, located between the restriction site NdeI and XhoI, and the recombinant plasmid was named pET-28a-BFD (such as figure 1 a). In addition, the mutants of BFD also have the function of catalyzing the synthesis of 2-hydroxyacetaldehyde from 1 formaldehyde. The mutants of BFD are listed in the Chinese invention patent application CN201710096307.X (publication number CN106916794A). GALS is a mutant of BFD. Its activit...

Embodiment 2

[0042] The expression of embodiment 2 gene

[0043] In order to detect the enzyme activity of GALS and FSA (A129T / A165G) in vitro, the enzyme was expressed and purified in Escherichia coli.

[0044] (1) The Escherichia coli expression recombinant plasmids pET-28a-GALS and pET-28a-FSA(A129T / A165G) were respectively transferred into E.coli BL21(DE3) to obtain recombinant bacteria. Positive clone selection (Kan+, 100 mg / mL) was carried out on a kanamycin-resistant plate, and cultured overnight at 37°C;

[0045] (2) Pick a single clone into 5 mL LB liquid medium (Kan+, 100 mg / mL), and culture at 37° C. and 220 rpm until the OD600 is 0.6-0.8. Transfer the bacterial liquid in 5mL LB medium to 800mL 2YT medium (Kan+, 100mg / mL), and cultivate to OD at 37°C and 220rpm 600 When the temperature is 0.6-0.8, cool down to 16°C, add IPTG to a final concentration of 0.5mM, and induce expression for 16h;

[0046] (3) The above-mentioned cultured bacteria liquid is collected in the bacteria ...

Embodiment 3

[0048] Example 3 protein purification

[0049] (1) Bacteria destruction: use a high-pressure and low-temperature crusher to destroy bacteria twice at a pressure of 1200 bar and 4°C. Centrifuge at 10000rpm for 45min at 4°C;

[0050] (2) Purification: the supernatant was suction-filtered through a 0.45 μm microporous membrane, and then purified by nickel affinity chromatography. The specific steps were as follows:

[0051] a: column balance: before hanging the supernatant, first use ddH 2 O washes 2 column volumes, then equilibrates 1 column volume of Ni affinity chromatography column with protein buffer;

[0052] b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5mL / min, repeat once;

[0053] c: Elution of impurity proteins: wash 1 column volume with protein buffer, and then use 50mL protein buffer containing 50mM and 100mM imidazole to elute strongly bound impurity proteins;

[0054] d: Elution of the target prot...

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

No PUM Login to View More

Abstract

The invention provides a method for biosynthesizing L-xylose. The method is characterized in that aldolase, such as D-fructose-6-phosphate aldolase (FSA), is used for converting substrates formaldehyde and glycolaldehyde into the L-xylose; or a combination of glycolaldehyde condensing enzyme (GALS), a mutant thereof or an enzyme with a function of catalyzing formaldehyde to synthesize glycolaldehyde, such as benzoyl formate decarboxylase (BFD), and aldolase, such as D-fructose-6-phosphate aldolase (FSA), can be utilized to convert the substrate formaldehyde into L-xylose by a one-pot method. The biosynthesis method disclosed by the invention has the advantages of high conversion rate, simple production process, greenness, friendliness, easiness in large-scale production and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for biosynthesizing L-xylose. Background technique [0002] Although rare sugars are rare in nature, they play an important role in the fields of diet, health care, and medicine because of their potential biological activity and low toxicity. Rare sugars usually have the sweetness of natural sugars, but they cannot or are rarely metabolized by the body, so they can be used as substitutes for high-calorie sugars such as sucrose. For example, xylitol, a pentose sugar substance, has a sweetness equivalent to 90% of sucrose , the heat is 60% of sucrose, can be used as an auxiliary therapeutic agent for diabetics, and has physiological activities such as lowering blood sugar, preventing dental caries, improving liver function, losing weight, laxative, and regulating intestinal function. In addition, rare sugars can inhibit the generation of active oxygen. ...

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): C12P19/02C12P7/24C12N9/88
CPCC12P19/02C12P7/24C12N9/88C12Y401/01007C12Y401/02013
Inventor 江会锋逯晓云刘玉万初斋林崔博卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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