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A method for biosynthesizing d-tagatose with immobilized enzyme catalyst

An immobilized enzyme and biosynthesis technology, applied in the field of bioengineering, can solve the problems of low conversion rate of D-tagatose, poor stability, low activity of L-arabinose isomerase, etc., and achieve improved conversion rate and efficient production. , the effect of improving vitality and stability

Active Publication Date: 2020-05-01
镇江百泰生物科技有限公司
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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects existing in the existing immobilized enzyme biosynthesis D-tagatose technology, such as low conversion rate from substrate D-galactose to D-tagatose, L-arabinose isomerase activity low, poor stability, etc., the invention provides a method for biosynthesizing D-tagatose with an L-arabinose isomerase immobilized enzyme catalyst, optimizing the biotransformation process of immobilized enzymes to synthesize D-tagatose, and realizing food Stable and efficient production of grade D-tagatose

Method used

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  • A method for biosynthesizing d-tagatose with immobilized enzyme catalyst

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Experimental program
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Embodiment 1

[0030] Spore Display L-AI Recombinant Vector and Construction of Recombinant Spore

[0031] 1. According to the characteristics of the multiple cloning site on the araA gene sequence of Lactobacillus brevis and the carrier pHS (no antibiotic gene), use bioinformatics software to design synthetic primers: primer 1, 5'- GGTGGCGGCGGTTCTGGTGGCGGCGGTTCT CTAAAGGAGTGCTCAATTATG-3'; Primer 2, 5'- CGCTCTAGAACTAGT CCATTTTAAATGTTTAGC. The sequence of the underlined part in primer 1 encodes Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (Linker), and the sequence of the underlined part in primer 2 is linear with EcoRI and BamHI double digestion One end of the pHS plasmid is complementary.

[0032] 2. Use Lactobacillus brevis genomic DNA as a template to amplify the target gene by PCR.

[0033] 3. PCR reaction parameters: pre-denaturation, 95°C for 5min; denaturation at 94°C for 2min, annealing, 55°C for 30s, extension, 72°C for 90s, after 35 cycles, keep at 72°C for 10min. Finally, keep war...

Embodiment 2

[0045] Conversion of 20g / L D-galactose to D-tagatose with L-arabinose isomerase immobilized enzyme catalyst

[0046] 1. Resuspend the recombinant Bacillus subtilis spores in phosphate buffer solution with pH 6.5 to make a concentration of 0.78×10 9 The recombinant Bacillus subtilis spore suspension of spore / mL is standby; Then 40mL (0.78×10 9 Spores / mL) Recombinant Bacillus subtilis spore suspension was added in 400mL reaction solution;

[0047] 2. Under the conditions of 70°C and 220rpm, shake and transform in a 2L shake flask for 24 hours, take samples from time to time, and obtain D-tagatose in the transformation liquid;

[0048] 3. Centrifuge at 8000rpm and collect the recombinant Bacillus subtilis spores in step 2 and add them to 400mL reaction solution;

[0049] 4. Repeat steps 2 and 3 4 times in sequence to calculate the conversion rate and residual activity of the immobilized enzyme catalyst.

[0050] Wherein the reaction liquid composition described in step 1 is as...

Embodiment 3

[0054] Conversion of 150g / L D-galactose to D-tagatose with L-arabinose isomerase immobilized enzyme catalyst

[0055] 1. Mix 40mL (0.78×10 9 Spores / mL) Recombinant Bacillus subtilis spore suspension was added in 400mL reaction solution;

[0056] 2. Under the conditions of 70°C and 220rpm, shake and transform in a 2L shake flask for 24 hours, take samples from time to time, and obtain D-tagatose in the transformation solution;

[0057] 3. Centrifuge at 8000rpm and collect the recombinant Bacillus subtilis spores in step 2 and add them to 400mL reaction solution;

[0058] 4. Repeat steps 2 and 34 in sequence to calculate the conversion rate and residual activity of the immobilized enzyme.

[0059] Wherein the composition of the reaction solution described in step 1 is as follows: D-galactose 150g / L, MnCl 2 1mM, 100mM pH6.5 potassium phosphate buffer;

[0060] Wherein the enzyme activity assay method described in step 4 is as follows: in 1mL reaction volume, contain 500mM D-...

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Abstract

The invention belongs to the technical field of bioengineering and specifically relates to a method for biosynthesis of D-tagatose by an immobilized enzyme catalyst. The method for biosynthesis of theD-tagatose by the immobilized enzyme catalyst is as follows: first, recombinant Bacillus subtilis is prepared, and an L-arabinose isomerase immobilized enzyme catalyst is constructed; and recombinantBacillus subtilis spores are added to a D-galactose reaction solution for transformation by oscillation under certain conditions to obtain the D-tagatose. The novel food-safe immobilized enzyme catalyst is constructed, the biotransformation process for biosynthesis of the D-tagatose by the immobilized enzyme catalyst is optimized, the transformation rate is improved, the specific activity of theimmobilized enzyme catalyst is as high as 2.10* 10<-9> U / spore, and the transformation rate of the D-tagatose reaches 95%. A new green, safe, high-efficiency and economical D-tagatose synthesis technology and method are developed, the high-efficiency production of the food-grade D-tagatose is realized, and the method has broad industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for biosynthesizing D-tagatose with an immobilized enzyme catalyst, in particular to a method for converting D-galactose into D-tagatose with an L-arabinose isomerase immobilized enzyme catalyst Sugar method. Background technique [0002] D-tagatose is a rare ketohexose that exists in nature. It has the same taste as sucrose, and its sweetness is 92% of that of sucrose. The U.S. Food and Drug Administration (FDA) has confirmed its food safety (GRAS ). As a low-calorie sweet prebiotic, D-tagatose is widely used in fruit juice, beverages, chewing gum and other food fields instead of sucrose, and has the effects of improving intestinal flora, treating type 2 diabetes, lowering cholesterol, and preventing colon cancer; , D-tagatose has potential effects on the treatment of diseases such as anemia, and can also be used for antioxidant and cell protection, and has a w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/02C12N11/16C12R1/125
CPCC12N9/90C12N11/16C12P19/02C12Y503/01003
Inventor 齐向辉张欢欢员君华杨苗苗朱婧斐田纳张国艳
Owner 镇江百泰生物科技有限公司
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