公开(公告)号：US20080070287A1

公开(公告)日：2008-03-20

申请号：US11852274

申请日：2007-09-07

申请人： Jorn Soe ,  Jorn Mikkelson ,  Arno Kreij

发明人： Jorn Soe ,  Jorn Mikkelson ,  Arno Kreij

IPC分类号： C12N5/04 ,  A21D13/04 ,  A23L1/32 ,  C12P7/64 ,  C12N9/10 ,  C12N15/00

CPC分类号： C12P7/6463 ,  C12P7/6454

摘要： The present invention relates to a method for the production of a variant lipid acyltransferase comprising the steps of: (i) selecting a parent enzyme which is a lipid acyltranserase enzyme; (ii) modifying one or more amino acids to produce a variant lipid acyltransferase; (iii) testing the activity of the variant lipid acyltransferase on a galactolipid and/or phospholipid and/or triglyceride substrate; (iv) selecting a variant enzyme with enhanced activity towards galactolipids compared with the parent enzyme; (v) providing a Bacillus licheniformis cell; (vi) transforming the Bacillus licheniformis cell with a heterologous nucleotide sequence encoding said variant lipid acyltransferase; and (iii) expressing said variant lipid acyltransferase in the cell under the control of a promoter sequence. The variant lipid acyltransferase can undergo post-translations modification, truncation and/or clipping, i.e., to remove a signal peptide. In addition, the present invention further relates to the use of Bacillus licheniformis to express a lipid acyltransferase, a Bacillus licheniformis host cell comprising a heterologous lipid acyltransferase and a vector comprising a nucleotide sequence encoding a lipid acyltransferase operably linked to a promoter sequence homologous to B. licheniformis.

摘要翻译： 本发明涉及生产变体脂质酰基转移酶的方法，包括以下步骤：（i）选择脂质酰基转移酶的亲本酶; （ii）修饰一个或多个氨基酸以产生变体脂质酰基转移酶; （iii）测试变体脂质酰基转移酶对半乳糖脂和/或磷脂和/或甘油三酯底物的活性; （iv）与亲本酶相比，选择具有增强的对半乳糖脂的活性的变体酶; （v）提供地衣芽孢杆菌细胞; （vi）用编码所述变体脂质酰基转移酶的异源核苷酸序列转化地衣芽孢杆菌细胞; 和（iii）在启动子序列控制下的细胞中表达所述变体脂质酰基转移酶。 变体脂质酰基转移酶可经历翻译后修饰，截短和/或剪切，即去除信号肽。 此外，本发明还涉及地衣芽孢杆菌表达脂质酰基转移酶，包含异源脂质酰基转移酶的地衣芽孢杆菌宿主细胞的使用和包含编码脂质酰基转移酶的核苷酸序列的载体，所述核苷酸序列可操作地连接到与B同源的启动子序列 地衣芽孢杆菌

公开(公告)号：US20050196766A1

公开(公告)日：2005-09-08

申请号：US10911160

申请日：2004-08-02

申请人： Jorn Soe ,  Jorn Mikkelson ,  Arno de Kreij

发明人： Jorn Soe ,  Jorn Mikkelson ,  Arno de Kreij

IPC分类号： C12N9/10 ,  C12N15/09 ,  C12Q1/68

CPC分类号： C12N9/1029 ,  A21D8/042 ,  A23L15/25 ,  A23L33/10 ,  A23L33/195 ,  C11B3/003 ,  C12P7/62 ,  C12P7/6436 ,  C12P7/6454 ,  C12P7/6481

摘要： The present invention relates to a method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme. The present invention further relates to variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

摘要翻译： 本发明涉及一种生产变体脂质酰基转移酶的方法，包括：（a）选择脂质酰基转移酶的母体酶，其特征在于该酶包含氨基酸序列基序GDSX，其中X是一个或多个 以下氨基酸残基L，A，V，I，F，Y，H，Q，TN，M或S; （b）修饰一个或多个氨基酸以产生变体脂质酰基转移酶; （c）测试变体脂质酰基转移酶对半乳糖脂底物和任选的磷脂底物和/或任选的甘油三酯底物的活性; （d）与母体酶相比，选择对半乳糖脂增强活性的变体酶; 和（e）制备一定量的变体酶。 本发明还涉及变体脂质酰基转移酶，其特征在于该酶包含氨基酸序列基序GDSX，其中X是以下氨基酸残基中的一个或多个L，A，V，I，F，Y，H，Q ，TN，M或S，并且其中当与SEQ ID No.2对应时，所述变体酶与任何一个或多个以下氨基酸残基的亲本序列相比，包含一个或多个氨基酸修饰：Ser3，Leu17，Ala114， Trp111，Tyr117，Leu118，Pro156，Gly159，Gln160，Asn161，Pro162，Ser163，Ala164，Arg165，Ser166，Gln167，Lys168，Val169，Val170，Glu171，Ala172，Tyr179，Gln182，Lys187，His180，Asn181，Met209，Leu210， Arg211，Asn215，Lys284，Met285，Gln289，Val290，Ala309，Ser310，Lys22，Met23，Gly40，Asn80，Pro81，Lys82，Val112，Asn87，Asn88,318。