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In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coli, the recombinant engineering strain was obtained. Afterwards, IPTG(isopropy-β-D-thiogalactopyranoside)was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. The results were as follows:(1) the amplified chlorophyllase gene AtCLH1 constructed the expression vector p ET28 a-At CLH1 successfully, obtaining the recombinant engineering strain;(2) induced under 30 ℃ for 22 h, the strain could well express the recombinant protein At CLH1 with 0.5 mmol/L IPTG, and the molecular weight was about 35 k Da;(3) the strain showed good chlorophyllase producing capability, and the activity of the produced chlorophyllase could reach up to 24.9 U/m L, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco;(4) based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach17.06% under the temperature of 37 ℃ at the humidity of 75% for 48 h;(5) after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.
In order to effectively reduce the chlorophyll content in flue-cured tobacco, improve the overall quality of tobacco leaves, chlorophyllase gene was cloned from Arabidopsis thaliana. After the expression of the expression vector in E. coli, the recombinant engineering strain was obtained. Afterwards , IPTG (isopropy-β-D-thiogalactopyranoside was used to induce the goal protein, and the chlorophyllase activity of the recombinant engineering strain was measured, so as to investigate its degradation effect on the chlorophyll in the extracts of tobacco leaves. were induced as follows: (1) the amplified chlorophyllase gene AtCLH1 constructed the expression vector p ET28 a-At CLH1 successfully, obtaining the recombinant engineering strain; (2) induced under 30 ° C for 22 h, the strain could well express the recombinant protein At CLH1 with 0.5 mmol / L IPTG, and the molecular weight was about 35 k Da; (3) the strain showed good chlorophyllase producing capability, and the activity of the produ ced chlorophyllase could reach up to 24.9 U / m L, which could degrade the chlorophyll in tobacco extract and had a good application prospect in improving the quality of low quality tobacco; (4) based on the results of orthogonal test, the enzyme extract from the strain was added to the tobacco leaf surface, which could make the degradation rate of chlorophyll in the tobacco leaf reach 17.06% under the temperature of 37 ° C at the humidity of 75% for 48 h; (5) after treated by the enzyme liquid, the test tobacco showed increase in the content of aromatic substances, enhancement of tobacco fragrance quality and amount, significant decrease of offensive odor and irritation, significant improvement of agreeable aftertaste, making the overall sensory quality of the tobacco leaf significantly improved.