A new type of amylase
A proteolytic enzyme and sequence technology, applied in the field of amylase, can solve problems such as deficiencies
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Embodiment 1
[0088] Embodiment 1: the preparation of amylase
[0089] The sequence shown in SEQ ID NO: 1 (the amino acid sequence encoded by it is shown in SEQ ID NO: 2) was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and cloned on the expression vector pET24a(+).
[0090] The pET24a(+) with the nucleotide sequence was transformed into Escherichia coli BL21(DE3) for inducible expression (refer to the third edition of "Molecular Cloning Experiment Guide", Science Press 2002, [US] J. Sambrook, Written by D.W. Russell, translated by Huang Peitang, etc.). The specific process is as follows:
[0091] Positive transformants were picked and seeded overnight at 37°C in LB medium. The seed culture solution was inoculated into the expression medium at an inoculum size of 1%, and cultivated at 37°C and 220 rpm until OD600=0.6-0.8.
[0092] After cooling to 16°C, add IPTG to 1mM for induction, and express overnight at 16°C and 190rpm. After induction of expression, the cells were col...
Embodiment 2
[0094] Example 2: OD value and starch content correlation standard curve formulation
[0095] Original iodine solution: Weigh 11.0g iodine and 22.0g potassium iodide, dissolve iodine completely with a small amount of water, and set the volume to 500ml.
[0096] Dilute iodine solution: Take 2.0ml of the original iodine solution, add 20g of potassium iodide, dissolve it in water and dilute to 500ml.
[0097] Prepare 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4 mg / ml starch solutions (dissolved in pH 7.5 phosphate buffer), add dilute iodine solution at a volume ratio of 1:5, measure and record the mixture OD660 value, make a correlation straight line, the linear relationship is as follows Figure 7 As shown, the corresponding formula is obtained: n=1.6111OD-0.0939.
Embodiment 3
[0098] Embodiment 3: Utilize the decline of iodine solution color to indicate the activity of amylase degrading starch
[0099] The enzyme activity of BM4 was measured by iodine-starch colorimetry. Specifically, the principle that starch turns blue when it encounters iodine is used for testing, see Guo Jing, Wang Yongjun, "Detecting the Activity of Industrial α-amylase by Domestic Dyed Starch Tablet Method", "Science of Daily Chemicals", 2000, 23(6):39–40.
[0100] Prepare 1.5g / l starch solution: weigh 1.5g starch and dissolve in 1L phosphate buffer.
[0101] Take 1ml of starch solution, preheat in a constant temperature water bath at 60°C for 8min, add 100μl of enzyme solution, and react for 5min accurately. Add dilute iodine solution at a ratio of 1:5. Measure and record the OD660 value of the mixture, and at the same time use the inactivated amylase enzyme solution as a control.
[0102] Enzyme activity definition: 1ml of liquid enzyme liquefies 1mg of starch in phospha...
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