CN101831467A - Method for preparing L-alanine by adding laurate alcohol ester phenylacetate - Google Patents
Method for preparing L-alanine by adding laurate alcohol ester phenylacetate Download PDFInfo
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- CN101831467A CN101831467A CN 201010152164 CN201010152164A CN101831467A CN 101831467 A CN101831467 A CN 101831467A CN 201010152164 CN201010152164 CN 201010152164 CN 201010152164 A CN201010152164 A CN 201010152164A CN 101831467 A CN101831467 A CN 101831467A
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- alcohol ester
- ala
- enzymatic production
- alanine
- aspartic acid
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Abstract
The invention discloses a method for preparing L-alanine by adding laurate alcohol ester phenylacetate, which comprises the steps of strain culture, fermentation enzyme production and enzymatic conversion reaction, product extraction and refining, wherein the fermentation enzyme production comprises the following steps: inoculating activated strains to a fermentation enzyme production culture medium of 0.01 to 0.2 volume percent of laurate alcohol ester phenylacetate with pH of 7.0, and culturing the strains for 24 hours at the temperature of 30 DEG C to obtain enzyme solution; and the enzymatic conversion reaction comprises the following steps: adding 0.01 to 0.2 volume percent of laurate alcohol ester phenylacetate into the enzyme solution, and performing the enzymatic conversion reaction under the conditions that the substrate is L-aspartic acid, the pH value is between 5.0 and 7.5 and the temperature is between 30 and 45 DEG C to generate the L-alanine. The method can improve the effective enzyme activity of L-aspartic acid-beta-decarboxylase and the reaction stability of the enzymatic reaction by using the laurate alcohol ester phenylacetate, and increase single bath feeding amount.
Description
Technical field
The present invention is a kind of method that toluylic acid bay alcohol ester prepares the L-L-Ala of adding, and belongs to biochemical engineering and technical field of enzyme engineering.
Background technology
The L-L-Ala (L-Alanine) also claim α-An Jibingsuan, white odorless crystalline powder.Be one of amino acid that life entity is interior with carbohydrate metabolism is closely related, demand is more, in industries such as medicine, food and chemical industry, be used widely, it is the important additives of food, makeup, fodder production, also be the main raw material of medicinal intermediates such as vitamin B6, calcium pantothenate, amino acid nutrient transfusion and other organic synthesis product, as antioxidant, phagostimulant, weedicide.It produces the another important amino acid industry that has become after L-glutamic acid, Methionin.
Abroad, Ri Ben the early start large-scale production L-of aginomoto company L-Ala.Method is also by extraction method. chemical synthesis, and developing into L-aspartic acid (L-Asp) is raw material, the biological respinse method that adopts L-aspartic acid-beta-decarboxylase to transform is produced.The employing propionic acid is that the chemical synthesis yield of raw material is low, and product purity is poor, and technology falls behind, and has serious contaminative.The biological respinse method is that raw material gets through enzymic transformations with the L-aspartic acid, and a complete set of equipment mainly partly is made up of microbial bacteria somatic cell culture, enzymic transformations, extraction refining system etc.This method facility investment is few, and operational path is short, and pollution-free, cost is low, and easy handling causes research and investor's concern and gradually adopts and become main flow.
In the biological respinse method, system enzyme cost has occupied the larger specific gravity of production cost.And to have determined to have a large amount of L-aspartic acid-beta-decarboxylases be to exist endobacillaryly owing to produce the characteristic of bacterial classification, i.e. this so-called intracellular enzyme partly is not utilized aborning and wastes.In the laboratory, when handling somatic cells as usefulness ultrasonic wave, high-pressure homogenization crush method etc., cell walls is destroyed, and enzyme discharges, and enzyme activity significantly improves.But existing production specifications can't adopt this type of wall breaking technology, therefore how to improve the system enzymatic process, improve effective extracellular enzyme vigor, and reducing system enzyme and production cost is the existing improved emphasis direction of production technique.
Toluylic acid bay alcohol ester is higher alcohols, hydrophobic, weakly water-wet thread-like molecule by force, is a kind of nonionogenic tenside, can significantly reduce solvent surface tension and liquid-liquid interface tension force, is a kind of good dispersant, emulsifying agent, organizes modifying agent and lubricant.Water-soluble under the low temperature, good froth breaking is arranged, press down the bubble effect, and nontoxic, nonirritant.
Summary of the invention
The purpose of this invention is to provide a kind of method that toluylic acid bay alcohol ester prepares the L-L-Ala of adding, can improve the stability of effective enzyme activity of L-aspartic acid-beta-decarboxylase and enzymic transformations, increase single batch of charging capacity.
The present invention achieves the above object by following technical solution:
A kind ofly add the method that toluylic acid bay alcohol ester prepares the L-L-Ala, comprise the steps:
1) spawn culture
To buy preserving number from Chinese Research for Industrial Microbial Germ preservation administrative center is 20147 pseudomonas (Pseudomonas dacunhae) bacterial classification, inserts the slant medium of pH7.0, cultivates 24h under 30 ℃ of conditions, obtains the activatory bacterial classification.Described slant medium is for containing peptone 2.0g, extractum carnis 1.0g, yeast extract paste 1.0g, sodium-chlor 1.0g, agar 4.0g and water 200ml.
2) enzymatic production and enzymic transformations
Above-mentioned activatory bacterial classification is inserted in the enzymatic production substratum of pH 7.0, cultivate 24h under 30 ℃ of conditions, obtain fermented liquid, i.e. enzyme liquid.Described enzymatic production substratum is for containing fumaric acid 30g, glucose 30g, peptone 17g, corn steep liquor 13g, KH
2PO
40.6g, MgSO
40.4g, toluylic acid bay alcohol ester 0.2-4.0ml and water 2000ml.
In above-mentioned enzyme liquid, add toluylic acid bay alcohol ester 0.2-4.0ml, substrate L-aspartic acid 6000-9000g, pH value is 5.0-7.5, temperature is to carry out generation L-L-Ala crude product enzymic transformations 5-7 days under 30-45 ℃ the condition.
When bacterial classification produces enzyme or enzymatic reaction, add a certain amount of toluylic acid bay alcohol ester, can significantly improve the stability of L-aspartic acid-beta-decarboxylase extracellular enzyme vigor and enzymic transformations, increase the actual service efficiency of enzyme, thereby improve single batch of charging capacity.
3) extraction is refining
Above-mentioned L-L-Ala crude product is added water 6000ml, and it is molten that stirring is warming up to 80 ℃ of weights, adds the 60g activated carbon decolorizing, filter, and 70 ℃ of concentrating under reduced pressure, crystallization is separated out in cooling, centrifugation, washing is drying to obtain L-L-Ala product.
Described toluylic acid bay alcohol ester is an analytical reagent.
The invention has the beneficial effects as follows:
1, toluylic acid bay alcohol ester consumption is few, cheap, can improve effective enzyme activity of L-aspartic acid-beta-decarboxylase and enzymatic reaction stability, increases single batch of charging capacity.
2, do not need to change original operational path and equipment, addition manner is simple, and is easy to operate, easily uses.
Embodiment
By the following examples technical scheme of the present invention is further described:
Reference examples
1) spawn culture
To buy preserving number from Chinese Research for Industrial Microbial Germ preservation administrative center is 20147 pseudomonas (Pseudomonas dacunhae) bacterial classification, inserts the slant medium of pH7.0, cultivates 24h under 30 ℃ of conditions, obtains the activatory bacterial classification.Described slant medium is for containing peptone 2.0g, extractum carnis 1.0g, yeast extract paste 1.0g, sodium-chlor 1.0g, agar 4.0g and water 200ml.
2) enzymatic production and enzymic transformations
Above-mentioned activatory bacterial classification is inserted in the enzymatic production substratum of pH 7.0, cultivate 24h under 30 ℃ of conditions, obtain fermented liquid, i.e. enzyme liquid.Described enzymatic production substratum is for containing fumaric acid 30g, glucose 30g, peptone 17g, corn steep liquor 13g, KH
2PO
40.6g, MgSO
40.4g, water 2000ml.
Add substrate L-aspartic acid 5300g in the above-mentioned enzyme liquid, pH value is 5.0-7.5, and temperature is to carry out enzymic transformations, stirring reaction 5 days, generation L-L-Ala crude product under 30 ℃ the condition.
3) extraction is refining
Above-mentioned L-L-Ala crude product is added water 6000ml, and it is molten that stirring is warming up to 80 ℃ of weights, adds the 60g activated carbon decolorizing, filter, and 70 ℃ of concentrating under reduced pressure, crystallization is separated out in cooling, centrifugation, washing is drying to obtain L-L-Ala product.
Embodiment 1
1) spawn culture
To buy preserving number from Chinese Research for Industrial Microbial Germ preservation administrative center is 20147 pseudomonas (Pseudomonas dacunhae) bacterial classification, inserts the slant medium of pH7.0, cultivates 24h under 30 ℃ of conditions, obtains the activatory bacterial classification.Described slant medium is for containing peptone 2.0g, extractum carnis 1.0g, yeast extract paste 1.0g, sodium-chlor 1.0g, agar 4.0g and water 200ml.
2) enzymatic production and enzymic transformations
Above-mentioned activatory bacterial classification is inserted in the enzymatic production substratum of pH 7.0, cultivate 24h under 30 ℃ of conditions, obtain fermented liquid, i.e. enzyme liquid.Described enzymatic production substratum is for containing fumaric acid 30g, glucose 30g, peptone 17g, corn steep liquor 13g, KH
2PO
40.6g, MgSO
40.4g, toluylic acid bay alcohol ester 0.2ml and water 2000ml.
Add toluylic acid bay alcohol ester, the substrate L-aspartic acid 6630g of 0.2ml in above-mentioned enzyme liquid, pH value is 5.0-7.5, and temperature is to carry out enzymic transformations, stirring reaction 5 days, generation L-L-Ala crude product under 30 ℃ the condition.
3) extraction is refining
Above-mentioned L-L-Ala crude product is added water 6000ml, and it is molten that stirring is warming up to 80 ℃ of weights, adds the 60g activated carbon decolorizing, filter, and 70 ℃ of concentrating under reduced pressure, crystallization is separated out in cooling, centrifugation, washing is drying to obtain L-L-Ala product.
Embodiment 2
1) spawn culture
To buy preserving number from Chinese Research for Industrial Microbial Germ preservation administrative center is 20147 pseudomonas (Pseudomonas dacunhae) bacterial classification, inserts the slant medium of pH7.0, cultivates 24h under 30 ℃ of conditions, obtains the activatory bacterial classification.Described slant medium contains peptone 2.0g, extractum carnis 1.0g, yeast extract paste 1.0g, sodium-chlor 1.0g, agar 4.0g and water 200ml.
2) enzymatic production and enzymic transformations
Above-mentioned activatory bacterial classification is inserted in the enzymatic production substratum of pH 7.0, cultivate 24h under 30 ℃ of conditions, obtain fermented liquid, i.e. enzyme liquid.Described fumaric acid 30g, glucose 30g, peptone 17g, corn steep liquor 13g, KH
2PO
40.6g, MgSO
40.4g, toluylic acid bay alcohol ester 1.0ml and water 2000ml.
Add toluylic acid bay alcohol ester, the substrate L-aspartic acid 7720g of 1.0ml in above-mentioned enzyme liquid, pH value is 5.0-7.5, and temperature is to carry out enzymic transformations, stirring reaction 6 days, generation L-L-Ala crude product under 37 ℃ the condition.
3) extraction is refining
Above-mentioned L-L-Ala crude product is added water 6000ml, and it is molten that stirring is warming up to 80 ℃ of weights, adds the 60g activated carbon decolorizing, filter, and 70 ℃ of concentrating under reduced pressure, crystallization is separated out in cooling, centrifugation, washing is drying to obtain L-L-Ala product.
Embodiment 3
1) spawn culture
To buy preserving number from Chinese Research for Industrial Microbial Germ preservation administrative center is 20147 pseudomonas (Pseudomonas dacunhae) bacterial classification, inserts the slant medium of pH7.0, cultivates 24h under 30 ℃ of conditions, obtains the activatory bacterial classification.Described slant medium is for containing peptone 2.0g, extractum carnis 1.0g, yeast extract paste 1.0g, sodium-chlor 1.0g, agar 4.0g and water 200ml.
2) enzymatic production and enzymic transformations
Above-mentioned activatory bacterial classification is inserted in the enzymatic production substratum of pH 7.0, cultivate 24h under 30 ℃ of conditions, obtain fermented liquid, i.e. enzyme liquid.Described enzymatic production substratum is for containing fumaric acid 30g, glucose 30g, peptone 17g, corn steep liquor 13g, KH
2PO
40.6g, MgSO
40.4g, toluylic acid bay alcohol ester 4.0ml and water 2000ml.
Add toluylic acid bay alcohol ester 4.0ml, substrate L-aspartic acid 8640g in above-mentioned enzyme liquid, pH value is 5.0-7.5, and temperature is to carry out enzymic transformations, stirring reaction 7 days, generation L-L-Ala crude product under 45 ℃ the condition.
3) extraction is refining
Above-mentioned L-L-Ala crude product is added water 6000ml, and it is molten that stirring is warming up to 80 ℃ of weights, adds the 60g activated carbon decolorizing, filter, and 70 ℃ of concentrating under reduced pressure, crystallization is separated out in cooling, centrifugation, washing is drying to obtain L-L-Ala product.
In the foregoing description, the input of substrate L-aspartic acid, the output of product L-L-Ala, molar yield, extract yield result are as shown in table 1:
Table 1
| Index | Reference examples | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| L-aspartic acid input amount (g) | ??5300 | ??6630 | ??7720 | ??8640 |
| L-L-Ala quantum of output (g) | ??3540 | ??4300 | ??5058 | ??5720 |
| The real L-L-Ala (g) that gets | ??3190 | ??3960 | ??4450 | ??5205 |
| L-L-Ala molar yield | ??97％ | ??97％ | ??98％ | ??99％ |
| L-L-Ala extract yield | ??90％ | ??92％ | ??90％ | ??91％ |
Annotate:
Claims (1)
1. one kind is added the method that toluylic acid bay alcohol ester prepares the L-L-Ala, comprises that spawn culture, enzymatic production and enzymic transformations, product extract and purification step, is characterized in that the actual conditions of described enzymatic production and enzymic transformations is:
Enzymatic production
The activatory bacterial classification is inserted in the enzymatic production substratum of pH 7.0, cultivate 24h under 30 ℃ of conditions, obtain fermented liquid, i.e. enzyme liquid, described enzymatic production substratum is for containing fumaric acid 30g, glucose 30g, peptone 17g, corn steep liquor 13g, KH
2PO
40.6g, MgSO
40.4g, toluylic acid bay alcohol ester 0.2-4.0ml and water 2000ml,
Enzymic transformations
In above-mentioned enzyme liquid, add toluylic acid bay alcohol ester 0.2-4.0ml, substrate L-aspartic acid 6000-9000g, pH value is 5.0-7.5, temperature is to carry out generation L-L-Ala crude product enzymic transformations 5-7 days under 30-45 ℃ the condition.
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| CN 201010152164 CN101831467A (en) | 2010-04-21 | 2010-04-21 | Method for preparing L-alanine by adding laurate alcohol ester phenylacetate |
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| CN 201010152164 CN101831467A (en) | 2010-04-21 | 2010-04-21 | Method for preparing L-alanine by adding laurate alcohol ester phenylacetate |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320480A (en) * | 2013-06-25 | 2013-09-25 | 南京大学 | Method for preparing beta-alanine by coupled enzymatic reaction |
| CN103387502A (en) * | 2012-05-09 | 2013-11-13 | 上海医药工业研究院 | Method for extracting L-alanine from L-alanine fermentation liquid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5327792B1 (en) * | 1974-03-21 | 1978-08-10 | ||
| CN101054570A (en) * | 2007-04-09 | 2007-10-17 | 南京工业大学 | Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell |
-
2010
- 2010-04-21 CN CN 201010152164 patent/CN101831467A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5327792B1 (en) * | 1974-03-21 | 1978-08-10 | ||
| CN101054570A (en) * | 2007-04-09 | 2007-10-17 | 南京工业大学 | Preparation for L-homophenylalanine by immobilization recombination of E.coliBL21-pET/aspC cell |
Non-Patent Citations (3)
| Title |
|---|
| 《工程科技I辑》 20090315 朱进伟 微生物法发酵生产L-精氨酸的研究 , 第3期 2 * |
| 《广西大学学报(自然科学版)》 19980315 黄时海等 表面活性剂对酶法转化生产丙氨酸的影响 , 第01期 2 * |
| 《生物加工过程》 20081115 贾红华等 化学-酶法制备L-高苯丙氨酸 第6卷, 第06期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103387502A (en) * | 2012-05-09 | 2013-11-13 | 上海医药工业研究院 | Method for extracting L-alanine from L-alanine fermentation liquid |
| CN103387502B (en) * | 2012-05-09 | 2015-03-11 | 上海医药工业研究院 | Method for extracting L-alanine from L-alanine fermentation liquid |
| CN103320480A (en) * | 2013-06-25 | 2013-09-25 | 南京大学 | Method for preparing beta-alanine by coupled enzymatic reaction |
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Application publication date: 20100915 |