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Bacterial strain of high-yielding acidic glycoside hydrolases

A glycoside hydrolase and acidic technology, applied in the field of microbiology, can solve the problems of poor understanding of acidophilus and limited acidophilus

Inactive Publication Date: 2013-04-17
EAST CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, the research on acidophilic bacteria is still very limited, only the physiological, biochemical and molecular genetic characteristics of a few kinds of bacteria have been studied, and little is known about many acidophilic bacteria that play an important role in nature

Method used

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  • Bacterial strain of high-yielding acidic glycoside hydrolases
  • Bacterial strain of high-yielding acidic glycoside hydrolases
  • Bacterial strain of high-yielding acidic glycoside hydrolases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 strain isolation

[0019] YF liquid medium (300mL): 8ml HBS (50×) (basic salt solution); 0.08g yeast extract; 0.12g Fructose; 292ml distilled water; 0.4ml trace elements solution; pH 3.0 (H 2 SO 4 ).

[0020] Enrichment medium (g / L): (NH 4 ) 2 SO 4 3.0, KCl 0.1, K 2 HPO 4 0.5, MgSO 4 ·7H 2 O 0.5, Ca(NO 3 ) 2 0.01, FeSO 4 ·7H 2 O 3.0, glucose 2, pH 2.0-2.5.

[0021] Separation medium (g / L): (NH 4 ) 2 SO 4 2.0, KCl 0.1, MgSO4 7H 2 O 0.25, Ca(NO 3 ) 2 0.01, glucose 1.0, yeast extract 0.1, pH 2.5-3.0.

[0022] LB medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, prepared with distilled water, with natural pH.

[0023] Take 20mL of the tail liquid of uranium leaching microorganisms in Jiangxi uranium mine, add it to 80mL enrichment medium for 72 hours at 30°C and 160rpm, shake and culture, pick a single colony and transfer them to YF medium and culture for two generations under the same conditions, repeat in parallel 3 times. The enri...

Embodiment 2

[0025] Embodiment 2 bacterial strain liquid fermentation produces enzyme analysis

[0026] Fermentation medium (g / L): (NH 4 ) 2 SO 4 2.0,K 2 HPO 4 1.0,; MgSO 4 ·7H 2 O 0.5; wheat bran: okara = 1:1 (total 1%), pH 3.0.

[0027] DNS method: The specific method is as follows: at pH 4.0, 50°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of glue beans, react for 10 minutes, add 1.5 mL of DNS to terminate the reaction, and boil for 5 minutes. After cooling to room temperature, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

[0028] pNPG method: substrate p-nitrophenyl β-D glucoside (pNPG) or p-nitrophenyl-β-galactopyranoside, p-nitrophenol α-D galactopyranoside was dissolved in 250uL at a concentration of 2mM Add 250uL of appropriately diluted enzyme solution to pH 4.0 buffer, react at 50°C for 10min, the...

Embodiment 3

[0030] The optimum pH of different enzymes of embodiment 3

[0031] Separation and purification of α-galactosidase, β-galactosidase, β-glucosidase, and mannanase, and performing enzymatic reactions at different pHs to determine their optimum pH. The substrates were tested for enzyme activity in 0.1mol / L citric acid-disodium hydrogen phosphate buffer solution with different pH at 50°C. result( figure 2) shows that the optimal pH of α-galactosidase is 4.0, and there is more than 80% relative enzyme activity at pH 3.5~6.5. The pH of β-galactosidase is 4.0, and it has more than 50% relative enzyme activity at pH 3.0~7.0. The optimal pH of β-glucosidase is 3.0, and there is more than 60% relative enzyme activity at pH 2.5~6.0. The optimal pH of mannanase is 3.5, and there is more than 40% relative enzyme activity at pH 3.0~7.0. The above results indicated that the optimal pH of the glycoside hydrolase produced by strain RBS-4 was in the acidic range, and it had higher activity...

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Abstract

The invention relates to the field of microbiology, in particular to a bacterial strain of high-yielding acidic glycoside hydrolases. The preservation number of the bacterial strain of the high-yielding acidic glycoside hydrolases is CGMCC NO. 5986. The invention aims to solve the technical problem of isolation of microbes which prefer acidic environment in prior art firstly, and provides the bacterial strain capable of producing acid enzymes. The bacterial strain provided by the invention is identified as a mycobacterium, and the optimum pH is 3.0, the produced enzymes have higher enzyme activity when the pH is within 3.0 to 4.0; and the bacterial strain can be used in industrial production needing acidic environment.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a bacterial strain with high acid glycoside hydrolase production. Background technique [0002] In plant cell walls, cellulose and hemicellulose are the most abundant renewable biomass resources in nature; their structure and composition are very complex. However, this important renewable resource has been difficult to be effectively utilized. Especially in the feed industry, since the gastrointestinal tract of animals is at an acidic pH, it is necessary to add a large amount of acid glycoside hydrolase from microbial sources, such as cellulase, xylanase, mannanase and other compound enzymes for effective feed utilization. [0003] Acidophilus is an extremely important class of microorganisms, widely distributed in acid mine wastewater, sulfur-containing hot springs and soil. Among them, the pH value of acid mine wastewater is usually below 3, and contains high concentrations of i...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/38C12N9/40C12N9/42C12R1/32
Inventor 李江刘亚洁吕飞龙王学刚孙占学徐玲玲史维俊徐蔚云石鹏君姚斌
Owner EAST CHINA UNIV OF TECH
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