sequences shown in fig1 the new sequence is aligned to the present alignment in fig1 by using the gap alignment to the most homologous sequence found by the gap program gap is provided in the gcg program package program manual for the wisconsin package version 8 august 1994 genetics computer group 575 science drive madison wis usa 53711 needleman s b and wunsch c d 1970 journal of molecular biology 48 443 45 the following settings are used for polypeptide sequence comparison gap creation penalty of 3 0 and gap extension penalty of 0 1 variants of a lipolytic enzyme can be obtained by methods known in the art such as site directed mutagenesis random mutagenesis or localized mutagenesis e g as described in wo 9522615 or wo 0032758 thermostable variants of a given parent lipolytic enzyme can be obtained by the following standard procedure streaking out on another assay plate u2014 secondary screening 1 degree higher then primary screening the following assay method is used to screen lipolytic enzyme variants and identify variants with improved thermostability 0072 e coli cells harboring variants of a lipolytic enzyme gene are prepared e g by error prone pcr random mutagenesis or localized random mutagenesis or by a combination of beneficial mutants and saturation mutagenesis the assay is performed with filters on top of a lb agar plate e coli cells are grown on cellulose acetate filters supplied with nutrients from the lb agar plate and under the selection pressure of ampicillin supplied with the lb agar proteins including the desired enzyme are collected on a nitrocellulose filter between lb agar and cellulose acetate filter this nitrocellulose filter is incubated in a buffer of desired ph generally 6 0 and at the desired temperature for 15 minutes e g 78 degrees for.