[发明专利]热稳定性提高的脂肪酶突变体

权利要求书

1.热稳定性提高的脂肪酶突变体，其特征在于由亲本华根霉(Rhizopuschinensis)CCTCC M 201021脂肪酶进行定向进化，通过易错PCR、DNAShuffling和定点突变，获得脂肪酶突变体，亲本华根霉氨基酸序列SEQ ID NO：1中在氨基酸取代位置发生突变，采用“原始氨基酸-位置-替换的氨基酸”来表示脂肪酶突变体中突变的氨基酸，所述脂肪酶突变体为：，

脂肪酶突变体氨基酸取代位置

突变体1       Ser234Phe

突变体2       Pro168Leu/Val329Ala

突变体3       Cys160Leu

突变体4       His317Pro/Met101Thr

突变体5       Ser151Asn/Leu180His

突变体6       Val261Gly/Ser373Cys

突变体7       Asp182Tyr/Ala230Phe

突变体8       Leu180His/Thr218Ser

突变体9       Asn366Asp/Leu180His/Val329Ala

突变体10      Ser234Phe/Leu180His/Thr218Ser

突变体11      Ser234Phe/Pro168His/Val329Ala/Cys160Leu/His317Pro/Met101Thr

突变体12      Leu180His/Thr218Ser/Val261Gly/Lys161Arg

突变体13      Glu107Gly/Ala129Ser/Glu363Arg/Lys161Arg/Val261Gly/Leu180His

突变体14      Lys161Arg/Leu180His/Thr218Ser/Met101Thr/Thr183Ala/

              Glu107Gly/Ser151Asn/Glu363Arg

突变体15      Glu107Gly/Ala129Ser/Ser151Asn/Lys161Arg/Leu180His/

              Ser234Phe/Glu363Arg/Val329Ala

突变体12-1    Ala230Phe/Leu180His/Thr218Ser/Val261Gly/Lys161Arg

突变体13-1    Glu107Gly/Ala129Ser/Ser151Asn/Lys219Asp/Leu180His/

              Glu363Arg/Ala230Phe

华根霉脂肪酶氨基酸突变体的氨基酸序列为SEQ ID NO：2，有22个突变氨基酸；

用于表达所述的脂肪酶突变体的表达载体为：pPIC9K，pPIC3.5K，pPICZα，pPICZ；

用于所述的表达载体转化的微生物宿主细胞为毕赤酵母。

2.根据权利要求1所述的脂肪酶突变体，其特征在于与亲本华根霉脂肪酶相比，所述脂肪酶突变体的热稳定性获得了提高，以在65℃下的半衰期t₅₀来表示热稳定性的提高，所述脂肪酶突变体的半衰期以及提高的倍数如下：

脂肪酶突变体   氨基酸取代位置                    65℃t₅₀(min)    65℃t₅₀(min)

                                                                 提高倍数

突变体1        Ser234Phe                         35.7            2.18

突变体2        Pro168Leu/Val329Ala               36.1            2.19

突变体3        Cys160Leu                         17.0            1.03

突变体4        His317Pro/Met101Thr               19.1            1.16

突变体5        Ser151Asn/Leu180His               21.7            1.32

突变体6        Val261Gly/Ser373Cys               33.5            2.03

突变体7        Asp182Tyr/Ala230Phe               27.9            1.69

突变体8        Leu180His/Thr218Ser               25.6            1.55

突变体9        Asn366Asp/Leu180His/Val329Ala     55.4            3.36

突变体10       Ser234Phe/Leu180His/Thr218Ser     89.9            5.45

突变体11       Ser234Phe/Pro 168His/Val329Ala/   329.0           19.94

               Cys160Leu/His317Pro/Met101Thr

突变体12       Leu180His/Thr218Ser/Val261Gly/    410.3           24.87

               Lys161Arg

突变体13       Glu107Gly/Ala129Ser/Glu363Arg/    465             28.19

               Lys161Arg/Val261Gly/Leu180His

突变体14       Lys161Arg/Leu180His/Thr218Ser/    450             27.27

               Met101Thr/Thr183Ala/Glu107Gly/

               Ser151Asn/Glu363Arg

突变体15       Glu107Gly/Ala129Ser/Ser151Asn/    399             24.19

               Lys161Arg/Leu180His/Ser234Phe/

               Glu363Arg/Val329Ala

突变体12-1     Ala230Phe/Leu180His/Thr218Ser/    755             45.76

               Val261Gly/Lys161Arg

突变体13-1     Glu107Gly/Ala129Ser/Ser151Asn/    690             41.82

               Lys219Asp/Leu 180His/Glu363Arg/

               Ala230Phe

亲本华根霉脂肪酶在65℃下的半衰期t₅₀为16.5min。