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Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion

A technology of cysteine ​​and enzymatic conversion, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of large amount of enzyme source cells, affecting technology industrialization, and low product production, etc. Achieve the effects of improved reaction efficiency, good transformation activity and improved efficiency

Active Publication Date: 2012-03-07
天津世纪伟康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In the above transformation reaction studies, the cultured cells of the TS-1138 strain were used as the enzyme source. The amount of the enzyme source cells was large (250 g / L), and the amount of the product produced was relatively small, and the enzyme source cells had to be prepared on the spot, because they were stable. Poor performance and cannot be stored for a long time, resulting in inconvenient use and affecting the industrialization of this technology

Method used

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  • Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion
  • Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion
  • Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation and Enzyme Activity Analysis of TS1138 Bacterial Cells

[0027] Pick a ring of TS-1138 strains from the plate and insert it into the liquid seed medium (20 grams of glucose, 3 grams of ATC, 5 grams of corn steep liquor, 3 grams of urea, 1.5 grams of NaCl, MnSO 4 ·H 2 O 0.1 g, K 2 HPO 4 3 g, MgSO 4 ·7H 2 O 0.5 g, FeSO 4 ·7H 2 (0.01 gram, constant volume to 1 liter, pH 7.5), 28 ℃, 200 turn and cultivate for 16 hours, then transfer in the enzyme production medium with 1% inoculum size (glucose 20 grams, ATC 4 grams, corn steep liquor 1 g, urea 3 g, NaCl 1.5 g, MnSO 4 ·H 2 O 0.1 g, K 2 HPO 4 3 g, MgSO 4 ·7H 2 O 0.5 g, FeSO 4 ·7H 2 O 0.01 g, constant volume to 1 liter, pH7.5), 28 ° C, 200 rpm for 16 hours, the resulting fermentation broth was centrifuged at 6,000 rpm at 4 °C for 10 minutes, the bacteria were collected, and buffered with 50 mmol / L of phosphate buffer solution (pH 8.0) to suspend and wash, then centrifuge, and collect the b...

Embodiment 2

[0029] The selection of embodiment 2 penetration reagent

[0030] Make the bacterial cells into 10% bacterial suspension with 50 mmol / L phosphate buffer (pH 8.0), add different osmotic reagents (see Table 1), shake at 30°C for 30 minutes, and rotate at 6,000 rpm at 4°C Centrifuge for 10 minutes to collect the cells, wash with 50 mmol / L phosphate buffer (pH 8.0) and centrifuge to obtain permeabilized cells. The enzyme activity of the permeabilized cells was tested according to the method in Example 1 to determine the best permeation reagent.

[0031] Table 1 Enzyme activity of permeabilized cells obtained after treatment with different permeabilization reagents

[0032]

[0033] After the TS-1138 cells were infiltrated with the penetrant, the ability of converting DL-ATC to generate L-cysteine ​​is shown in Table 2. Among them, the penetration effect of 2% toluene on the TS-1138 cells was better than that of other penetrants. The enzyme activity of the obtained permeabiliz...

Embodiment 3

[0035] The influence of embodiment 3 osmotic treatment temperature and time on enzyme activity

[0036] The appropriate temperature range and treatment time for infiltration were measured respectively with the same amount of bacteria, and the results were as follows: figure 2 As shown, taking the enzyme activity of non-permeabilized cells as a control, the enzyme activity of permeabilized cells obtained by treating at 20-35° C. for 15-90 minutes all increased by more than 2 times. However, in a lower temperature and a shorter time range (such as 20-25°C treatment for 15-60 minutes), the enzyme activity of the permeabilized cells increased with the extension of the infiltration time; when the infiltration time continued to increase, the enzyme activity The decline may be due to the leakage of some intracellular enzymes (such as 90 minutes at 20°C). In the case of higher temperature, such as 35 ° C after 15 minutes of treatment, the enzyme activity began to decrease, indicatin...

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Abstract

The invention provides preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion. The preparation method is characterized in that toluene is taken as a penetrating agent to treat bacterial suspension to obtain a TS-1138 strain permeabilized cell and the enzyme activity of the TS-1138 strain permeabilized cell is as high as 229.9U / g, about 7 times that of the initial cell; in a freeze-dried permeabilized cell, sodium glutamate, glycerine and mannitol are adopted as compound freeze-drying protective agents, the highest enzyme activity of theresiduals after freeze-drying is 66.7%, and after the residuals are stored at 4 DEG C for 3 months, the enzyme activity can be still over 90%; and in a spray-dried cell, sodium glutamate, Arabic gumsor beef extracts are adopted as the protective agents, the highest enzyme activity of the residuals after spray-drying is 68.92%, the activity of the prepared spray-dried enzyme powder is 614.7U / g and the enzyme powder can be stably stored for 3 months after being sealed at 4 DEG C. The solid microbial enzyme preparation obtained by the invention has high activity, is beneficial to storage and can be used for industrial production for preparing L-cysteine through enzyme method.

Description

【Technical field】 [0001] The invention uses microbial solid enzyme preparations to produce amino acids through enzymatic conversion, and belongs to the field of biotechnology. It relates to osmotic treatment and freezing or spray drying of microbial cells, and its application in transforming and producing L-cysteine. 【Background technique】 [0002] L-cysteine ​​is an important sulfur-containing amino acid, widely used in medicine, food, cosmetics and feed industries. At present, the production of L-cysteine ​​in my country is mainly based on the hydrolysis of hair, which has low output and high energy consumption. During the hydrolysis process, a large amount of waste gas and waste acid are produced, which pollutes the environment. The enzymatic preparation of L-cysteine ​​has the advantages of mild reaction conditions, strong specificity, high efficiency, less by-products, and environmental friendliness. It is of great significance to energy saving, emission reduction, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/12C12R1/40
Inventor 高智慧张奇刘磊陈晓芸张玺姚瑞娟
Owner 天津世纪伟康生物科技有限公司