A process for producing an optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane (2), comprising a step of asymmetric hydrolysis of 2-alkyl-1,1,3-trialokoxycarbonylpropane (1) by using an enzyme capable of selectively hydrolyzing an ester moiety of either one enantiomer of 2-alkyl-1,1,3-trialkoxycarbonylpropane (1), or by using a culture of a microorganism capable of producing the enzyme or a treated object thereof.

The invention provides methods of synthesizing a viral protease inhibitor in high yield, without using expensive catalysts or challenging reaction conditions.

Provided are a lipase mutant and an application thereof. Specifically, one or more mutations selected from A262H, A338V, V3641, A158PN, and 1159N are generated on the basis of an amino acid sequence as shown in SEQ ID NO: 1; and compared with a parental lipase, a change in the structure and function of a protein occurs in the lipase mutant, and the stereoselectivity is improved, such that a usage amount of an enzyme is decreased to a certain extent, the post-treatment difficulty is reduced, and the lipase mutant is suitable for industrial production.

The present invention provides a method for producing an atropisomer of a pyrrole derivative having excellent mineralocorticoid receptor antagonistic activity, and an intermediate thereof. A method for producing an atropisomer of a pyrrole derivative using a compound represented by (B) [wherein R.sup.1 represents a C1-C4 alkyl group, and R.sup.2 represents a 2-hydroxyethyl group or a carboxymethyl group] as a production intermediate.

##STR00001##

A process for producing an optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane (2), comprising a step of asymmetric hydrolysis of 2-alkyl-1,1,3-trialokoxycarbonylpropane (1) by using an enzyme capable of selectively hydrolyzing an ester moiety of either one enantiomer of 2-alkyl-1,1,3-trialkoxycarbonylpropane (1), or by using a culture of a microorganism capable of producing the enzyme or a treated object thereof.

This invention refers to the obtainment of a modified lipolytic enzyme that was isolated, expressed and purified from the heterologous expression. The gene sequence that codifies for the basal enzyme was obtained based on a thermo acidophilus organism of the acidobacteraceae family. This basal enzyme that comes from a thermo acidophilus organism, it is able to hydrolyze lipid substrates (triacylglycerols) united to middle chain fatty acids (C.sub.6-C.sub.10) such as tributyrine and tricapryln, among others. It also can carry out other inverse reactions to the hydrolysis such as synthesis reactions. On the other hand, this enzyme has enantioselective preference on (S) substrates of profens esters such as ibuprofen, naproxen and others. The enantioselective lipolytic basal enzyme was modified in its terminal C end to add an amino acid histidine tail that gives a higher efficiency in its purification process. The invention therefore refers to a method for making a pure, active polypeptide, which is called lipolytic enzyme 499EST obtained through the host E. coli BL 21 (DE3).

The present invention provides a novel hydrolase that can industrially produce optically highly pure (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid with high efficiency at low costs, and a production method using the hydrolase.

The invention belongs to the field of bioengineering and food technology, and discloses a method for enzymatic resolution of chiral substances, including the following steps: (1) preparing an enzyme solution with a lipase concentration of 1-3000 U/mL, and adding a soluble salt, a hydrophilic solvent and a hydrophobic solvent to the enzyme solution to form a three-liquid phase system; the hydrophobic solvent contains esters or amide compounds composed of racemic chiral compounds; (2) subjecting the three-liquid phase system to enzyme-catalyzed reaction under stirring condition; after the reaction is completed, standing or centrifuging the three-liquid phase system to divide it into three layers, which are a upper liquid layer, a middle liquid layer and a lower liquid layer from top to bottom. The optically pure chiral product after hydrolysis is mainly rich in the middle liquid layer or the lower liquid layer, while the upper liquid layer product is another ester or amide product containing an optically pure chiral product. The method has the advantages of low energy consumption, high raw material utilization rate, and mild reaction conditions, and solves the problems of low chiral resolution efficiency, poor chiral selectivity, low recovery rate, and difficulty in industrialization in the existing enzymatic method.

Provided are a method for preparing (R)-ketorolac and use thereof. The method includes the following steps: step 1, preparing ketorolac; and step 2, subjecting the ketorolac to resolution to obtain the (R)-ketorolac, the resolution being selected from the group consisting of chiral amine resolution and enzyme resolution.