Improve Lipase Gene ROL Expression Level Based On Rational Design And Gene Dose Effect

Rhizopus oryzae lipases for its perfect enzyme characteristics such as regioselectivity and stereoselectivity have potential application in the fields of biodiesel,food processing,organic synthesis,and chiral compound synthesis.On the other hand,due to the relatively complicated production process of lipase,the production cost is relatively high,and the ensuing disadvantage is to limit its development in the fields of biodiesel and so on.In order to highlight that enzymatic preparation of biodiesel is superior to chemical methods,it is more environmentally friendly and more competitive in the market.Using genetic engineering,molecular biology,protein engineering and other technical means,artificially construct a lipase genetically engineered strain with relatively high enzyme activity and relatively excellent protein expression.It seems particularly urgent and necessary.A lipase gene ROL derived from Rhizopus oryzae was artificially synthesized according to the original gene sequence in Gen Ban K(1LGY).The ROL gene expression,protein domain optimization and other technical methods were used to improve gene expression level.The high-efficiency expression Pichia pastoris recombinants were constructed,and their enzyme activity,protein content,and the enzyme production capacity were evaluated.The main works carried out were listed as following:1.The original ROL gene recombinant expression strain had the highest activity of 260 U/mL and the highest protein content of 0.14 mg/mL in the flask cultivation.After optimization,the enzyme activity and protein content were increased to 560 U/mL and 0.28 mg/mL.2.A multi-copy tandem was constructed for the optimized lipase gene.The average enzyme activity of single copy lipase ROL was 205 U/mL,the average enzyme activity of two copies of lipase ROL was 960 U/mL,and the average enzyme activity of three copies of recombinant ROL was 1,250 U/mL.The average protein content of single-copy lipase ROL,two-copy,and three-copy lipase ROL were 0.24 mg/mL,0.37 mg/mL,and 0.42 mg/mL,respectively.It can be seen that with the increase of gene dosage,the enzyme activity and protein content were enhanced.3.The enzyme production capacity of the optimal strain containing multi-copy ROL genes was tested in a 50 L bioreactor.Under optimized conditions,the highest activity was 26,500 U/mL.Compared with the optimal single-copy enzyme activity of the original gene,the enzyme activity was increased by 101 times and the protein content was increased by 20 times.Some of the above data can be reacted to a certain extent,and the necessary molecular transformation of foreign genes can be given to enzymes with new characteristics.Towards the direction expected by people,these technical methods include the accumulation of gene copy number,which will involve the construction of high-copy vectors.In addition,protein domain optimization can also be carried out,including the increase of protein stability.Considering that there are many factors that affect the expression of Pichia pastoris,a single optimization factor often has a limited effect in targeted transformation.Therefore,in actual operation,it is necessary to combine multiple technical methods to achieve the desired experimental effect.