Novel amylase
A phospholipase and sequence technology, applied in the field of new amylases, can solve problems such as deficiencies
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Embodiment 1
[0088] Example 1: Preparation of amylase
[0089] The sequence shown in SEQ ID NO: 1 (the encoded amino acid sequence is shown in SEQ ID NO: 2) was synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd., and cloned on the expression vector pET24a(+).
[0090] The pET24a(+) with the nucleotide sequence was transformed into E. coli BL21(DE3) for inducible expression (refer to the third edition of "Molecular Cloning Experiment Guide", Science Press 2002, [美]J. Sam Brook, Written by DW Russell, translated by Huang Peitang and others). The specific process is as follows:
[0091] Pick positive transformants in LB medium and conduct overnight seed culture at 37°C. Inoculate the seed culture solution into the expression medium at an inoculum amount of 1%, and cultivate it at 37°C and 220 rpm to OD600=0.6-0.8.
[0092] After cooling to 16°C, IPTG was added to 1mM for induction, and expression was carried out at 16°C and 190rpm overnight. After the induction of expression, the cells we...
Embodiment 2
[0094] Example 2: Development of a standard curve for the correlation between OD value and starch content
[0095] Original iodine solution: Weigh 11.0g iodine and 22.0g potassium iodide, use a small amount of water to completely dissolve the iodine, and dilute to 500ml.
[0096] Dilute iodine solution: absorb 2.0ml of original iodine solution, add 20g potassium iodide, dissolve in water and dilute to 500ml.
[0097] Prepare 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4 mg / ml starch solution (dissolved with 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 line, the linear relationship is as Figure 7 As shown, the corresponding formula is obtained: n=1.6111OD-0.0939.
Embodiment 3
[0098] Example 3: Use the decrease in color of iodine solution to indicate the activity of amylase to degrade starch
[0099] The enzyme activity of BM4 was measured by iodine-starch colorimetry. Specifically, the principle of using starch to turn blue when met with iodine can be found in Guo Jing and Wang Yongjun, "Domestic dyed starch tablet method to detect the activity of industrial alpha-amylase", "Science of Daily Use Chemicals", 2000, 23(6): 39-40.
[0100] Prepare 1.5g / l starch solution: weigh 1.5g starch and dissolve it in 1L phosphate buffer.
[0101] Take 1ml of starch solution, preheat it in a constant temperature water bath at 60℃ for 8min, add 100μl enzyme solution, and react accurately for 5min. Add diluted iodine solution in a ratio of 1:5. Measure and record the OD660 value of the mixed solution, and at the same time use the inactivated amylase enzyme solution as a control.
[0102] Enzyme activity definition: 1ml liquid enzyme liquefies 1mg starch under the condit...
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