Starch pullulanase with wide pH value adaptability and application

A technology of pullulanase and crude pullulanase, applied in the field of enzyme engineering and biomodified starch, can solve the problems of acid-base consumption, affecting the utilization of glucose syrup, etc.

Active Publication Date: 2020-10-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the subsequent saccharification reaction, the glucoamylase cannot hydrolyze the α-1,4-glucosidic bond between fructose and glucose, and there is a maltulose by-product in the product after saccharification, which will affect the subsequent glucose syrup production. use
In addition, the optimal reaction condition of glucoamylase is about pH4.5-5.0, so it is necessary to adjust the pH value of the liquefied product before the saccharification step, resulting in acid-base consumption

Method used

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  • Starch pullulanase with wide pH value adaptability and application
  • Starch pullulanase with wide pH value adaptability and application
  • Starch pullulanase with wide pH value adaptability and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the preparation of starch pullulanase

[0035] Synthesize the gene shown in SEQ ID NO.2, insert the gene of the starch pullulanase encoded by SEQ ID NO.2 into the plasmid pET-28a (+), construct the expression vector Amypul-pET-28a (+), and insert The expression vector was introduced into the host bacteria Escherichia coli E.coli BL21(DE3) for fermentation to produce enzyme. The seed solution was inoculated into LB medium containing 50 μg / mL kanamycin at an inoculum amount of 5%, and after shaking the flask at 37°C and 160 rpm for 70 minutes, the OD value fell between 0.6-0.8. Afterwards, inducer IPTG with a final concentration of 0.4 mM was added, and cultured at 18° C. and 160 rpm for 24 hours. The fermentation broth was centrifuged at 4°C and 8000×g for 15 minutes to obtain bacterial cells. The bacteria were subjected to ultrasonic disruption (240W), and then centrifuged at 4° C. and 8000×g for 30 minutes to obtain a crude enzyme solution. The crude e...

Embodiment 2

[0036] Embodiment 2: Enzyme activity and optimum pH, pH stability test of starch pullulanase

[0037] (1) Optimum pH determination of enzyme activity

[0038] Prepare buffers with pH values ​​of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 to replace the buffer in the enzyme activity assay of starch pullulanase at 75 The activity of starch pullulanase was measured at ℃, and the highest enzyme activity was defined as 100%, and the relative enzyme activity was calculated by comparing the rest of the enzyme activities, so as to investigate the optimal pH of the enzyme (see the test results in figure 2 ), the results showed that the starch pullulanase had the highest activity under acidic condition pH5.5 and alkaline condition pH8.5, and the activity was equal. In the range of pH 4.0-10.0, the enzyme shows stability, and the activity remains above 50%.

[0039] The optimal pH list of enzymes from other sources is shown in Table 1. It can be seen from the c...

Embodiment 3

[0045] Embodiment 3: the application of starch pullulanase in starch liquefaction

[0046] Prepare potato starch milk with a concentration of 30% (w / v), adjust the pH to 5.5, add 10U of starch pullulanase, heat up and liquefy, keep it at 75°C for 30min, measure the DE value of the reaction product, and the DE value is 14.2.

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Abstract

The invention discloses starch pullulanase with wide pH value adaptability and application, and belongs to the technical field of enzyme engineering and biologically modified starch. The invention provides a novel starch pullulanase which is used for starch liquefaction so as to provide a proper substrate for subsequent starch deep processing. The starch pullulanase has the same activity under theconditions of pH 5.5 and pH 8.5, and more than half of stable enzyme activity is kept between pH 4.5 and pH 10.0. The optimum temperature of the enzyme is 75-80 DEG C, and the half-life period of theenzyme is about 8 h. Due to the thermal stability and wide pH action range of the enzyme, the enzyme can be independently used or compounded with various enzymes, is applied to starch deep processing, improves the starch liquefaction efficiency and the utilization rate of a starch substrate, provides a new idea for starch processing, and has huge potential and application prospect.

Description

technical field [0001] The invention relates to a starch pullulanase with wide pH adaptability and its application, belonging to the technical field of enzyme engineering and bio-modified starch. Background technique [0002] At present, the amount of starch used for industrial transformation in the world is as high as 50 million tons per year, and 10 million tons of starch is used in my country every year for the conversion of glucose, sugar for fermentation or sugar for alcohol production. Sugar occupies an important position in the national economy because of its close relationship with the national economy and people's livelihood, and it is widely used in food, beverage, fermentation and other fields. At present, starch sugar production process is mainly enzymatic method, including two stages of liquefaction and saccharification. The liquefaction stage is the most important stage in the entire process of converting starch to glucose, and its results have a great impact ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/16C12P19/04C12N15/56C12N15/63C12N9/44
CPCC12P19/16C12P19/04C12N9/2457C12Y302/01041
Inventor 柏玉香李晓晓金征宇
Owner JIANGNAN UNIV
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