CN108220267A - 磷脂酶及其应用 - Google Patents
磷脂酶及其应用 Download PDFInfo
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- CN108220267A CN108220267A CN201611200242.0A CN201611200242A CN108220267A CN 108220267 A CN108220267 A CN 108220267A CN 201611200242 A CN201611200242 A CN 201611200242A CN 108220267 A CN108220267 A CN 108220267A
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Abstract
本发明涉及磷脂酶及其应用。具体而言,本发明提供一种多肽,所述多肽选自:(1)在SEQ ID NO:2所示氨基酸序列第182、221、274、323、339、342、369和500位中的至少一个位置上具有取代突变的多肽;和(2)由(1)所示的多肽与促进(1)所示多肽表达与纯化的多肽组成的多肽。本发明还提供编码该多肽的多核苷酸序列,含该多核苷酸序列的核酸构建物,宿主细胞,以及相关的用途。本发明的多肽为磷脂酶,可用于油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业中。
Description
技术领域
本发明涉及磷脂酶及其应用。
背景技术
磷脂酶(PL)是生物体内存在的能水解甘油磷脂的酶,根据磷脂酶水解甘油磷脂的位点(Richmond G.S.等,Int.J.Mol.Sci.,2011,12:588-612)不同,可将磷脂酶分为磷脂酶A1(PLA1)、磷脂酶A2(PLA2)、磷脂酶B(PLB)、磷脂酶C(PLC)、磷脂酶D(PLD)以及溶血磷脂酶A。
PLA1能够水解二脂肪酰基磷脂的Sn-1位酰基酯键,产生溶血磷脂和脂肪酸。PLA2则是能够水解Sn-2位的酰基酯键,产生溶血磷脂和脂肪酸。PLB则是能水解二脂肪酰基磷脂的Sn-1位和Sn-2位的两个酰基酯键产生甘油磷酰胆碱和游离脂肪酸的磷脂酶,该酶也同时具有溶血磷脂酶的活性。PLC是能够水解甘油骨架和磷酸基之间的磷酸酯键产生二磷脂酰甘油和磷酸极性醇酯(如磷酸胆碱、磷酸乙醇胺、磷酸肌醇等)。PLD则是水解磷酸基和氨基醇之间的磷酸酯键生磷脂酸和氨基醇。溶血磷脂酶A则是指能够水解溶血磷脂的Sn-1位或Sn-2位的脂肪酰基酯键,产物是甘油磷酸胆碱和脂肪酸。
磷脂酶广泛存在于各种生物体中,譬如人体中就有PLA2、PLB、PLC等多种类型的磷脂酶。因为微生物具有生产周期短、生长条件简单、生产效率高等特点,其一直是工业酶制剂领域研究的重点。
当前市场上在售的磷脂酶品种主要有Ultra、10L、 F、Oil、G999、A2、PLC等。其中Ultra为一种人工改造的蛋白,表现为PLA1的活性,其基因来源于疏棉状嗜热丝孢菌(Thermomyceslanuginosus)的一种脂肪酶和尖孢镰刀菌(Fusariumoxysporum)脂肪酶的杂交体。其磷脂酶的最适合作用温度在45℃附近,其脂肪酶的最适合作用温度在20℃。因此当温度高于40℃将以磷脂酶的活性为主,而脂肪酶的活性较低。是一种来自于镰刀菌属微生物Fusariumvenenatum的磷脂酶A1,其最适合作用温度为55℃。F的磷脂酶为一种来自于丝状真菌曲霉属的磷脂酶A2。Oil、10L和A2都是PLA2,前者的基因来源于紫红色链霉菌(Streptomyces violaceoruber),后两个磷脂酶商品的基因都是猪胰腺PLA2。G999为一种仅作用于溶血磷脂的溶血磷脂酶A,PLC为一种人工改造的磷脂酶C,其能够快速作用于磷脂酰胆碱和磷脂酰乙醇胺等底物,但是几乎不作用于磷脂酰肌醇和磷脂酸。
磷脂酶可广泛应用于油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业等多个方面(Demaria L.等,ApplMicrobiol.Biotechnol.,2007,74(2):290-300)。如CN1780908等公开报道了磷脂酶A可以用于烘烤、用于洗涤剂、改善水溶液或糖浆的过滤性以及制备溶血磷脂等。磷脂酶还可以应用于奶酪生产工艺中,其可以提高奶酪的得率(Nielsen P.H.等,Int J.Life Cycle Assess,2009,14:137–143.)。磷脂酶D可以用于制备高纯度的磷脂酰肌醇、磷脂酰丝氨酸以及利用磷脂酶D的碱基转移活性可将一些多肽、核苷和多糖类药物通过配位键连接在磷脂载体上,制成具有特殊疗效作用的脂质体等(陈石良等,《工业微生物》,1999,29(4):47-50)。
磷脂酶另外一个比较具有十分广阔应用前景的用途是在油脂精炼中,磷脂酶A1、A2、磷脂酶B以及磷脂酶C都可应用于酶法脱胶过程(Clausen K,Eur.J.Lipid Sci.Tech.,103(6),2001,333-340;Jiang F等,Bioresour.Technol.,2011,102(17):8052-8056;中国专利CN102634411、CN107455等)。
植物油毛油中的磷脂成分非常复杂,以大豆油的毛油为例,其中的磷脂成分有磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、磷脂酰肌醇(PI)、磷脂酸(PA)、溶血磷脂酰胆碱(LPC)、溶血磷脂酰乙醇胺(LPE)、溶血磷脂酸(LPA)、磷脂酰丝氨酸(PS)、甘油磷脂酰胆碱(PG)、双磷脂酰甘油(DPG)以及鞘磷脂(SM)等(Glonek T.JAOCS.,1998,75(5):569-573)。如此多的磷脂成分存在于胶质中,为了达到较好的脱胶效果就要求所使用的磷脂酶具有相对低的底物选择性,通常的PLA和PLB都基本满足此条件,而PLC则具有较高的底物选择性。
发明内容
本发明提供了一种磷脂酶的突变体,具体地,所述的磷脂酶突变体能够水解磷脂的脂肪酰基酯键,该磷脂酶突变体的磷脂酶比活性增加,且在50-55℃的脱胶温度下温度稳定性不下降,适合毛油脱胶等的应用。
因此,本发明第一方面提供一种多肽,所述多肽选自:
(1)在SEQ ID NO:2所示氨基酸序列第182、221、274、323、339、342、369和500位中的至少一个位置上具有取代突变的多肽;和
(2)由(1)所示的多肽与促进(1)所示多肽表达与纯化的多肽组成的多肽。
在一个或多个实施方案中,所述多肽的氨基酸序列如SEQ ID NO:13所示。
在一个或多个实施方案中,所述多肽的氨基酸序列如SEQ ID NO:6、8、10或12所示。
在一个或多个实施方案中,所述多肽为磷脂酶。
本发明第二方面提供一种多核苷酸,所述多核苷酸选自:
(1)编码本发明多肽的多核苷酸;
(2)与(1)所述的多核苷酸序列互补的多核苷酸;和
(3)(1)或(2)所述的多核苷酸的长10-40个碱基的片段。
在一个或多个实施方案中,所述多核苷酸的序列如SEQ ID NO:5、7、9或11所示。
本发明第三方面提供一种多核苷酸构建物,所述多核苷酸构建物含有本发明所述的多核苷酸。
在一个或多个实施方案中,所述多核苷酸构建物是表达载体或克隆载体。
本发明第四方面提供一种遗传工程化的宿主细胞,所述宿主细胞:
(1)表达本发明所述多肽;和/或
(2)含有本发明的多核苷酸或多核苷酸构建物。
本发明第五方面提供一种组合物,所述组合物含有本发明的多肽和任选的辅料,优选的,所述辅料为选自活性炭、氧化铝、硅藻土、多孔陶瓷、多孔玻璃的吸附材料。
本发明第六方面提供本发明所述氨基酸序列、多核苷酸序列、核酸构建物、遗传工程化的宿主细胞或组合物在油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业中的应用。
本发明第七方面提供一种酶法脱胶方法,包括混合本发明多肽与毛油进行脱胶的步骤。
在一个或多个实施方案中,所述脱胶包括在50~60℃下搅拌1~5小时,然后升温至80~90℃保持1~10分钟。
在一个或多个实施方案中,以毛油重量计,多肽的添加量为50~1000ppm,优选为100~500ppm,更优选为100~300ppm。
在一个或多个实施方案中,在将多肽加入毛油前,先将毛油加热至50~70℃,优选50~60℃。
在一个或多个具体实施例中,所述毛油包括但不限于:大豆油、葵花籽油、花生油、菜籽油、米糠油、玉米油、橄榄油、棕榈油、棕榈仁油、棕榈软脂、卡诺拉油、蓖麻油、椰子油、芫荽油、棉籽油、榛子油、大麻籽油、亚麻籽油、芒果仁油、白芒花油、牛蹄油、红花油、山茶花油、妥尔油、椿油和其他植物油。
附图说明
图1:磷脂酶突变体库磷脂酶筛选图。其中,四张图各自为磷脂酶变体在筛选平板上的点板,其中每张图最中间一个菌为磷脂酶亲本,左上图中黑方块标记处为含磷脂酶变体154的菌,右上图中黑方块标记处为含磷脂酶变体2的菌,左下图中黑方块标记处为含磷脂酶变体10的菌,右下图中黑方块标记处为含磷脂酶变体17的菌。
图2:亲本磷脂酶和各变体磷脂酶的温度稳定性。
图3:亲本磷脂酶的磷脂酶活性的最适温度曲线。
图4:变体磷脂酶2的磷脂酶活性的最适温度曲线。
图5:亲本磷脂酶的最适pH曲线。
图6:变体磷脂酶2的最适pH曲线。
图7:金属离子对亲本磷脂酶活性的影响。
图8:金属离子对变体磷脂酶2活性的影响。
具体实施方式
本发明提供了一个或多个磷脂酶突变体,本发明还包括这些突变体的表达载体构建,变体磷脂酶的制备方法及变体磷脂酶的温度稳定性等。本发明所提供的亲本磷脂酶和变体磷脂酶多肽具有能够水解二脂肪酰基磷脂的Sn-1,2位的磷脂酶活性,分子量约为25KDa,该亲本磷脂酶及变体磷脂酶不能水解三油酸甘油酯,没有脂肪酶活性,该亲本磷脂酶及变体磷脂酶可以在食品加工、油脂脱胶、医药等多个领域具有应用前景。
具体而言,本发明提供的多肽为在SEQ ID NO:2所示氨基酸序列第182、221、274、323、339、342、367和500位中的至少一个位置上具有取代突变的多肽。
用性能相近或相似的氨基酸进行保守性取代通常不会改变蛋白质或多肽的功能。“性能相近或相似的氨基酸”包括例如,具有相似侧链的氨基酸残基的家族,这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
或者,根据其化学结构,“性能相近或相似的氨基酸”包括脂肪族氨基酸,如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺;芳香族氨基酸,如苯丙氨酸和酪氨酸;杂环族氨基酸,如组氨酸和色氨酸;和杂环亚氨基酸,如脯氨酸。
因此,在某些实施方案中,第182位的保守性取代可以是异亮氨酸被选自丙氨酸、缬氨酸、亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺中的一种氨基酸取代;或者,所述保守性取代可以是异亮氨酸被选自丙氨酸、缬氨酸、亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸中的一种氨基酸取代。
在某些实施方案中,第221位上的保守性取代可以是谷氨酰胺被选自甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸和半胱氨酸中的一种氨基酸取代;或者可以是谷氨酰胺被选自丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸和天冬酰胺中的一种氨基酸取代。
在某些实施方案中,第274位上的保守性取代可以是天冬氨酸被选自谷氨酸或丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺中的一种氨基酸取代。
在某些实施方案中,第323位上的保守性取代可以是谷氨酸被选自天冬氨酸或丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺的一种氨基酸取代。
在某些实施方案中,第339位上的保守性取代可以是亮氨酸被选自丙氨酸、缬氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸中的一种氨基酸取代;或者被选自丙氨酸、缬氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺中的一种氨基酸取代。
在某些实施方案中,第342位上的保守性取代可以是异亮氨酸被选自丙氨酸、缬氨酸、亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺中的一种氨基酸取代;或者,所述保守性取代可以是异亮氨酸被选自丙氨酸、缬氨酸、亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸中的一种氨基酸取代。
在某些实施方案中,第367位上的保守性取代可以是精氨酸被选自赖氨酸、或组氨酸或丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺中的一种氨基酸取代。
在某些实施方案中,第500位上的保守性取代可以是丙氨酸被选自缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸中的一种氨基酸取代,或者被选自缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺中的一种氨基酸取代。
在某些实施方案中,用来取代第182位或第342位的异亮氨酸的氨基酸是与天冬酰胺属于同一氨基酸类别的氨基酸,包括甘氨酸、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸和半胱氨酸,以及丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸和精氨酸。
在某些实施方案中,用于取代第221位上的谷氨酰胺的氨基酸是与赖氨酸同属一类氨基酸类的氨基酸,包括精氨酸和组氨酸,以及丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺。
在某些实施方案中,用于取代第274位上的天冬氨酸的氨基酸是与谷氨酸同属一类氨基酸类的氨基酸,包括天冬氨酸,以及丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺。
在某些实施方案中,用于取代第323位上的谷氨酸的氨基酸是与甘氨酸同属一类氨基酸类的氨基酸,包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸和半胱氨酸,以及丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸和精氨酸。
在某些实施方案中,用于取代第339位上的亮氨酸的氨基酸是与苯丙氨酸属于同一氨基酸类别的氨基酸,包括酪氨酸、色氨酸和组氨酸;或者是与异亮氨酸属于同一氨基酸类别的氨基酸,包括丙氨酸、缬氨酸、亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸,以及甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺和谷氨酰胺。
在某些实施方案中,用于取代第342位上的异亮氨酸的氨基酸是与天冬酰胺属于同一氨基酸类别的氨基酸,包括甘氨酸、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸和半胱氨酸,以及丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸和精氨酸。
在某些实施方案中,用于取代第367位上的精氨酸的氨基酸是与色氨酸属于同一氨基酸类别的氨基酸,包括丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸,以及组氨酸。
在某些实施方案中,用于取代第500位上的丙氨酸的氨基酸是与苏氨酸属于同一氨基酸类别的氨基酸,包括甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、酪氨酸和半胱氨酸,以及丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸和精氨酸。
在某些实施方案中,本发明的多肽在选自第182、221、274、323、339、342、367和500位的至少2个或至少3个位置上具有所述取代。
在某些实施方案中,本发明的多肽至少在第339位上具有所述突变,并任选地在选自第182、274、323和500位的一个、两个或三个位置上具有所述突变。在某些实施方案中,所述多肽在第182、323和339位上存在所述突变。在其它实施方案中,所述多肽在第274、339和500位上存在所述突变。在某些实施方案中,所述多肽仅在第342位存在所述突变。在其它实施方案中,所述多肽在第221和367位上存在所述突变。
在某些实施方案中,本发明的多肽至少在第339位存在L339I或L339F突变,并任选地在选自第182、274、323和500位的一个、两个或三个位置上具有所述突变,优选地,182位上的突变为I182N,274位上的突变为D274E,323位上的突变为E323G,500位上的突变为A500T。在某些实施方案中,所述多肽在第182、323和339位上存在的突变为I182N、E323G和L339I或L339F。在其它实施方案中,所述多肽在第274、339和500位上存在的突变为D274E、L339I或L339F和A500T。在某些实施方案中,所述多肽仅在第342位存在I342N突变。在其它实施方案中,所述多肽在第221和367位上存在的突变为Q221R和R367W。
在某些实施方案中,本发明的多肽存在以下突变中的至少一个、至少两个或至少三个:I182N、Q221R、D274E、E323G、L339I或L339F、I342N、R367W和A500T。
本发明还包括在SEQ ID NO:13所示的氨基酸序列中除前述第182、221、274、323、339、342、367和500位外的其它位置上经过取代、缺失或添加一个或几个氨基酸,同时保留SEQ ID NO:6、8、10或12所具备的磷脂酶活性的由SEQ ID NO:13衍生的多肽。所述几个通常为10个以内,优选8个以内,更优选5个以内。
在知晓SEQ ID NO:6、8、10或12的序列和生物学功能的情况下,本领域技术人员可采用常规的技术手段判断SEQ ID NO:6、8、10或12所示的氨基酸序列中哪些氨基酸残基可被取代或删除。例如,通过比对来自不同种属、活性相同或类似或明显不同的序列,可以判断这些序列中哪些氨基酸残基是可以被取代或删除。可采用本领域常规的方法(包括本发明所公开的方法)验证这样的序列是否具备本发明所述的酶活。
本发明SEQ ID NO:2来自枝孢霉WBRD00050(CGMCC No.7508)。来自枝孢霉属(Cladosporium)其它株的与本发明SEQ ID NO:13具有至少95%、优选至少96%、优选至少97%、优选至少98%、优选至少99%的序列同一性的多肽也包括在本发明范围之内。优选地,来自以下属的微生物,且与本发明SEQ ID NO:13具有至少95%、优选至少96%、优选至少97%、优选至少98%、优选至少99%的序列同一性的多肽也包括在本发明范围之内:曲霉菌属(Aspergillus)、青霉属(Penicillium)、Graphiopsis属、Rachicladosporium属、Verrucocladosporium属、煤炱属(Capnodium)、Antennariella属、Conidioxyphium属、Fumiglobus属、Leptoxyphium属、Microxyphium属、Polychaeton属、Scorias属、锥梗孢属(Dissoconium)、乌氏霉属(Uwebraunia)、Rasutoria属、叠孢属(Microcyclospora)、小叠孢属(Microcyclosporella)、拟维朗那霉属(Pseudoveronaea)、枝氯霉属(Ramichloridium)、链丝孢属(Scleroramularia)、平脐疣孢属(Zasmidium)、月盾霉属(Peltaster)、接瓶霉属(Zygophiala)、链丝孢属(Scleroramularia)、球腔菌属(Mycosphaerella)、横断孢属(Strelitziana)、节担孢属(Wallemia)、派伦霉属(Peyronellaea),栓菌属(Trametes),单端孢属(Trichothecium)、Davidiella属、Rasutoria属、顶齿霉属Acrodontium、糙孢菌Asperisporium、尾孢菌属Cercospora、小尾孢菌属Cercosporella、球腔菌属(Mycosphaerella)、钉孢属(Passalora)、假尾孢菌属(Pseudocercospora)、假小尾孢菌属(Pseudocercosporella)、柱隔孢菌属(Ramularia)、壳针孢属(Septoria)、长蠕孢属(Helminthosporium)、木霉属(Trichoderma)、根霉属(Rhizopus)、毛霉属(Mucor)等丝状真菌。更优选的是,本发明的多肽来源于枝孢霉属(Cladosporium)的微生物。可采用本领域周知的方法计算两条序列之间的序列同一性,这些方法如采用软件BLAST。
此外,本领域技术人员公知,在基因克隆操作中,常常需要设计合适的酶切位点,这势必在所表达的蛋白末端引入了一个或多个不相干的残基,而这并不影响目的蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化,常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内,例如,包括但不限于,适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。本发明氨基酸序列的氨基端或羧基端还可含有一个或多个多肽片段,作为蛋白标签。任何合适的标签都可以用于本发明。例如,所述的标签可以是FLAG,HA,HA1,c-Myc,Poly-His,Poly-Arg,Strep-TagII,AU1,EE,T7,4A6,ε,B,gE以及Ty1。这些标签可用于对蛋白进行纯化。使用的标签例子包括Poly-Arg,如RRRRR(SEQ ID NO:14);Poly-His 2-10个(通常6个),如HHHHHH(SEQ ID NO:15);FLAG,即DYKDDDDK(SEQ ID NO:16);Strep-TagII,即WSHPQFEK(SEQ ID NO:17);和C-myc,即WQKLISEEDL(SEQ ID NO:18)。应理解,这些氨基酸序列的存在不会影响到所得多肽的活性。因此,本发明也包括在本发明多肽的C末端和/或N末端添加一个或数个氨基酸所得的多肽,这些多肽仍具有本文所述的磷脂酶活性。
根据重组生产方案所用的宿主,本发明的多肽可以是糖基化的,或可以是非糖基化的。
本发明的多肽可以是天然纯化的产物,或是化学合成的产物,或使用重组技术从原核或真核宿主(例如,细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。
本申请包括本发明多肽的编码序列。SEQ ID NO:5、7、9和11显示了本发明多肽的编码序列例子。所述“编码序列”包括与例如SEQ ID NO:5、7、9或11高度同源的序列或在严格条件下与例如SEQ ID NO:5、7、9或11杂交的核苷酸序列或与上述分子高度同源的家族基因分子。编码本发明多肽的序列可以与例如SEQ ID NO:5、7、9或11所示的编码区序列相同或者是简并的变异体。如本文所用,“简并的变异体”在本发明中是指编码相同的氨基酸序列但核苷酸序列有差别的核苷酸序列。
编码本发明多肽的序列包括:只编码成熟多肽的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
本发明的多肽的编码序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可先采用常规的技术手段从基因组DNA,然后再根据本发明所公开的核苷酸序列,尤其是开放阅读框序列来设计引物,用于从该基因组DNA中扩增出脂肪酶基因。
因此,本发明也包括本发明编码序列的片段,所述片段通常长10~40个碱基、优选长15~30个碱基,能作为引物或探针使用。“片段”在本文中指全长序列的连续的一部分序列。
本发明也涉及核酸构建物,该核酸构建物含有本发明的编码序列以及与该编码序列操作性连接并指导该编码序列在宿主细胞中在合适的条件下表达的一个或多个调控序列。编码本发明多肽的多核苷酸可以各种方式被操作,以保证该多肽的表达。在其插入载体之前该多核苷酸序列的操作可能根据该表达载体而是合乎需要或必需的。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
调控序列可以是合适的启动子序列,为用于表达编码本发明多肽的多核苷酸的宿主细胞识别的核苷酸序列。启动子序列包含接到多肽表达的转录调控序列。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列,包括突变的、截短的和杂合启动子,并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
用于指导本发明的核酸构建体,特别是在细菌宿主细胞中转录的合适启动子的实例是从噬菌体T7启动子、大肠杆菌lac操纵子、天蓝色链霉菌(Streptomyces coelicolor)琼脂糖酶基因、枯草芽孢杆菌果聚糖蔗糖酶基因、地衣芽孢杆菌α-淀粉酶基因、解淀粉芽孢杆菌α-淀粉酶基因、地衣芽孢杆菌青霉素酶基因等。
用于指导本发明的核酸构建体在丝状真菌宿主细胞正转录的合适启动子的实例是从米曲霉TAKA淀粉酶、米黑根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定的α-淀粉酶、黑曲霉或泡盛曲霉糖化酶(glaA)、里氏木霉纤维二糖水解酶I、氏木霉纤维二糖水解酶II、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、里氏木霉葡聚糖内切酶等基因获得的启动子及其突变、截短和杂合(hybrid)启动子。
在酵母宿主中,有用的启动子可获得自酿酒酵母烯醇酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母乙醇脱氢酶、3-磷酸甘油醛脱氢酶、酿酒酵母磷酸丙糖异构酶、酿酒酵母3-磷酸甘油酸激酶的基因、巴斯德毕赤酵母醇氧化酶基因。用于酵母宿主细胞的其它有用启动子由Romanos et al.,1992,Yeast 8:423-488描述。
调控序列也可以是合适的转录终止子序列,为由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3′末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。用于细菌宿主的优选终止子可以是来自T7噬菌体的终止子。用于丝状真菌宿主细胞的优选终止子获得自米曲霉TAKA淀粉酶、黑曲霉葡萄糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉α-葡萄糖苷酶的基因。用于酵母宿主细胞的优选终止子获得自酿酒酵母烯醇酶、酿酒酵母细胞色素C、酿酒酵母甘油醛-3-磷酸脱氢酶、巴斯德毕赤酵母醇氧化酶基因等。
调控序列也可以是合适的前导序列,对宿主细胞翻译重要的mRNA的非翻译区。前导序列与编码该多肽的核苷酸序列的5′末端可操作连接。在选择的宿主细胞中有功能的任何前导序列都可用于本发明。
调控序列也可以是编码与多肽的氨基酸末端连接的氨基酸序列并且指导该编码的多肽进入细胞分泌途径的信号肽编码区。核苷酸序列编码序列的5’端可固有地包含天然连接具有编码分泌多肽的编码区节段的翻译阅读框的信号肽编码区。备选地,编码序列的5’端可包含与该编码区外源的信号肽编码区。当编码序列非天然地包含信号肽编码区时,可能需要外来的信号肽编码区。备选地,外来的信号肽编码区可简单地替换天然的信号肽编码区以增强多肽的分泌。然而,指导表达的多肽进入选择的宿主细胞的分泌途径的任何信号肽编码区,即分泌进入培养基,都可用于本发明。
本发明也涉及包括本发明多核苷酸的克隆载体或表达载体。这些载体可含有前文所述的各调控序列。
表达载体可以是能够方便地经受重组DNA方法并且可导致感兴趣的核苷酸序列表达的任何载体(如质粒或病毒)。载体的选择一般取决于载体与其中被导入该载体的宿主细胞的相容性。该载体可以是线性或闭合的环形质粒。
载体可以是自主复制的载体,即作为染色体外实体存在,其复制不依赖于染色体复制的载体,例如质粒、染色体外元件、微型染色体或人工染色体。载体可包含用于保证自我复制的任何方式。备选地,载体可以是当被导入宿主细胞时,整合到基因组中并且与其已经被整合进入的染色体一起复制的载体。此外,可使用一起包含将被导入宿主细胞基因组的总DNA的单个载体或质粒或两个或多个载体或质粒,或转座子。
本发明的载体优选包含一个或多个容许容易选择转化、转染、转导等细胞的可选择标记。可选择的标记是基因,其产物提供对抗生素或病毒的抗性、对重金属的抗性、原养型至营养缺陷型等。
本发明的载体优选包含容许该载体整合进入宿主细胞基因组或该载体在细胞中独立于基因组自主个复制的元件。
一个以上拷贝的本发明的多核苷酸可被插入宿主细胞以增加该基因产物的产量。多核苷酸拷贝数的增加可通过将至少一个附加拷贝的序列整合进入宿主细胞基因组或通过包括可扩增的选择标记基因和该多核苷酸来获得,其中包含扩增拷贝的选择标记基因并且由此包含附加拷贝多核苷酸的细胞可通过在存在适当的选择剂时培养该细胞来筛选。
本发明的载体优选包含人工合成的一段序列,含有多个限制内切酶识别位点,能为外源DNA提供多种可插入的位置或插入方案。本发明的表达载体更为优选包含有连续6个组氨酸序列的小肽,有利于蛋白质的提取和纯化。
本发明的表达载体更为优选地选择可用于毕赤酵母中表达的载体。本发明的载体优选商品化的毕赤酵母中使用的载体如pPIC、pPICZ、pAO、pGAP或pGAPZ等一系列的载体。
含有本发明多核苷酸序列的克隆载体可用于复制足够多的目标质粒。因此,本发明的克隆载体带有较强的自我复制元件,如复制起始位点等。通常,本发明的克隆载体不具有表达元件。
本发明也涉及含有被用于重组生产多肽的本发明多核苷酸的重组宿主细胞。包括本发明多核苷酸的载体被导入宿主细胞以使得该载体如早先说明的作为染色体的组成部分或作为染色体外的自我复制载体来维持。宿主细胞的选择很大程度上取决于编码多肽的基因及其来源。
宿主细胞可以是植物细胞或单细胞微生物或非单细胞微生物。单细胞微生物例如革兰氏阳性细菌,包括但不限于芽孢杆菌细胞,例如,嗜碱芽孢杆菌、解淀粉芽孢杆菌、短芽孢杆菌、巨大芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌、凝结芽孢杆菌、嗜热脂肪芽孢杆菌和苏云金芽孢杆菌等;或链霉菌细胞,例如钱青紫链霉菌;或革兰氏阴性细菌,例如大肠杆菌和假单胞菌属。在优选的方面,细菌宿主是枯草芽孢杆菌、大肠杆菌、地衣芽孢杆菌、嗜热脂肪芽孢杆菌和大肠杆菌细胞。
宿主细胞也可以是真核生物,例如哺乳动物、昆虫、植物、酵母或真菌细胞。在优选的方面,宿主细胞是真菌细胞,如在此使用的“真菌”包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)、接合菌门(Zygomycota)以及卵菌门等。
在更优选的方面,宿主细胞是子囊菌门的细胞如酵母属(Saccharomyces)、毕赤酵母属(Pichia)、耶氏酵母属(Yarrowia)、假丝酵母属(Candida)以及Komagataella属等。
在最优选的方面,宿主细胞是巴斯德毕赤酵母(Pichia pastoris)、酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolytica)等。在另外最优选方面,宿主细胞是巴斯德毕赤酵母(Pichia pastoris)细胞。
可采用常规的转染方法将含本发明多核苷酸序列的核酸构建物转入宿主细胞中。转染通常分为瞬时转染和稳定转染。前者外源DNA/RNA不整合到宿主染色体中,因此一个宿主细胞中可存在多个拷贝数,产生高水平的表达,但通常只持续几天。稳定转染中,外源DNA既可以整合到宿主染色体中,也可能作为一种游离体存在。转染的技术手段包括化学转染和物理转染,前者如DEAE-葡聚糖法、磷酸钙法和人工脂质体法,后者如显微注射、电穿孔和基因枪等。
可通过磷脂酶活性筛选而后通过克隆表达等技术获得本发明多肽的氨基酸及编码多核苷酸序列信息。筛选的方法可以包括文库法、蛋白分离纯化等方法,本发明优选地采用多步层析相结合的分离纯化方法纯化得到具有磷脂酶活性的蛋白,而后再进一步通过活性电泳及酶谱分析的方法获得具体的有活性的蛋白条带。为了获得目的蛋白的序列信息,可以选择常规的化学测序方法以及质谱鉴定等方法。
在获得多肽的编码序列后,可采用如下方法生产本发明多肽,该方法包括:(a)在有助于生产多肽的条件下培养宿主细胞;以及(b)回收该多肽。
本发明的生产方法中,细胞可以利用本领域已知的方法在适于生产多肽的培养基中培养。例如,细胞可通过在实验室或工业发酵罐中进行的摇瓶培养和小规模或大规模的发酵(包括连续的、分批的、分批补料或固态发酵),在合适的培养基和容许该多肽表达和/或分离的条件下进行培养。培养发生在利用本领域已知的方法包括碳源和氮源和无机盐的合适培养基中。合适的培养基可获得自商业供应者或可根据公开的组合物来制备。如果该多肽分泌进入培养基,该多肽可从培养基直接回收。如果该多肽不分泌进入培养基,它可从细胞裂解物回收。
多肽可利用本领域已知的特异于该多肽的方法来检测。这些检测方法可包括特异抗体的使用,酶产物的形成,或酶底物的消失。例如,酶活性测定可如在此说明的用来测定多肽的活性。
本发明所描述的多肽可利用本领域已知的方法来回收。例如,多肽可通过常规方法,包括但不限于离心、过滤、超滤、萃取、层析、喷雾干燥、冷冻干燥、蒸发或沉淀等从培养基回收。
本发明的多肽可通过本领域已知的多种方法来纯化,包括但不限于色谱法(如离子交换、亲和性、疏水性、色谱聚焦、分子排阻),电泳法(例如等电聚焦)、差异溶解度(如盐析沉淀)、SDS-PAGE或萃取法以获得基本上纯的多肽。
本发明的磷脂酶可应用于油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业等多个方面,包括但不限于用于烘烤、用于洗涤剂、改善水溶液或糖浆的过滤性以及制备溶血磷脂等。本发明的磷脂酶还可以应用于奶酪生产工艺中。
本发明的多肽可以纯的酶制品的形式提供,也可以组合物的形式提供。组合物可以是粉末组合物,也可以是液体组合物,或糊状组合物。以组合物形式提供时,根据该含酶组合物的不同用途,该组合物可含有不同的辅料。可将本领域已知的辅料添加到本发明的组合物中,这类辅料包括但不限于山梨醇、山梨酸钾、苯甲酸甲酯、苯甲酸乙酯、蔗糖、甘露糖、海藻糖、淀粉、氯化钠、氯化钙等稳定剂或其他物质中的一种或多种。
本发明也提供一种使用本发明多肽进行油脂精炼、磷脂改性、改良饲料、食品制备、药品制备的方法,所述方法(以及本文中提及的使用本发明多肽的其它方法)优选在以下条件下进行:
(A)温度为37℃-70℃,优选为50±5℃;和/或
(B)pH为3.7-7.0,优选3.4-5.8,更优选4.5±0.5。
本发明还提供一种水解磷脂的方法,该方法包括使含磷脂的成分与本发明的多肽接触。优选的接触条件如上文(A)-(B)点所述。优选的磷脂是大豆磷脂。
本发明方法中使用的本发明多肽的量可根据实际情况加以确定。
更进一步的,本发明还提供一种酶法脱胶方法,所述方法包括混合本发明多肽与毛油进行脱胶的步骤。
通常,先将毛油加热至50~70℃,优选50~60℃,然后再加入本发明的多肽。
多肽(即本发明的磷脂酶)通常以水溶液的形式加入。以毛油重量计,多肽的添加量为50~1000ppm,优选为100~500ppm,更优选为100~300ppm。
脱胶条件一般包括:在50~60℃下搅拌1~5小时,然后升温至80~90℃保持1~10分钟。
适用于本发明方法脱胶的毛油包括但不限于大豆油、葵花籽油、花生油、菜籽油、米糠油、玉米油、橄榄油、棕榈油、棕榈仁油、棕榈软脂、卡诺拉油、蓖麻油、椰子油、芫荽油、棉籽油、榛子油、大麻籽油、亚麻籽油、芒果仁油、白芒花油、牛蹄油、红花油、山茶花油、妥尔油、椿油和其他植物油。
下文将以具体实施例的方式阐述本发明。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等,《分子克隆:实验室指南》(美国纽约州:冷泉港实验室出版社(Cold Spring Harbor Laboratory Press),1989)所述的条件,或按照制造厂商所建议的条件进行。对于试剂的用法和用量,除非另有说明,否则按照常规的用法和用量使用。除非另有说明,实施例中所用的化学试剂均购自上海国药,为分析纯级;本发明中的限制性内切酶都购自NEW ENGLAND BIOLABS。
实施例中所用到的磷脂酶活力检测方法如下进行:
用来源于大豆的磷脂酰胆碱为底物来测定该多肽的活力大小,该方法以大豆来源的磷脂酰胆碱(>98%,购自阿拉丁)为反应底物,通过本发明多肽的催化产生游离脂肪酸。而后采用游离脂肪酸定量检测试剂盒(Wako Pure Chemical Industries,Ltd,Osaka,Japan)进行定量。根据产生游离脂肪酸的量从而计算出酶的活性。酶活力单位定义为每分钟催化产生1微摩尔的游离脂肪酸为1个单位。
实施例1:本发明磷脂酶易错PCR
EPCR体系(50ul):
以SEQ ID NO:1为模版,以SEQ ID NO:3所示的序列为上游引物和SEQ ID NO:4所示序列为下游引物作为引物进行易错PCR。
实施例2:本发明磷脂酶突变体库的构建
使用1%的琼脂糖(购自AMRECSO)凝胶电泳进行分离纯化易错PCR产物目标条带,并使用美国OmegaBio-Tek公司的E.Z.N.ATM胶回收试剂盒进行目标条带的回收。胶回收产物用AvrⅡ和NotΙ-HF两种限制性内切酶进行酶切反应,而后用OmegaBio-Tek公司的E.Z.N.ATM的产物回收试剂盒回收酶切产物。同时进行pPIC9K载体的酶切及酶切产物回收。
各取适量的上述两种回收的酶切产物进行混合,加入一定量的连接酶和连接酶缓冲液后,放置于22℃水浴中反应2小时左右。
取出1根装有100μl DH5α感受态细胞,冰浴融化后,相应加入20μl的连接产物,冰浴30min。而后于42℃热激90秒后,立即放置于冰浴中1-2min后,往每管中加入880μl的LB培养基。而后于37℃,200rpm摇床进行预培养60min左右。而后于12000rpm离心3min后,去除部分清液,留约100ul清液,充分悬浮沉淀菌体后,取全部液体涂布于相应的含氨苄霉素(购自上海生工)的平板上,37℃培养过夜;
取出培养过夜的转化平板,用水洗下平板上的菌落,离心收集菌体,而后提取重组质粒,该质粒即为构建成功的表达载体,命名为ECSPLB-pPIC9K。
质粒线性化,用SacΙ和BUFFER 4线性化质粒。
线性化条件:86.0μlECSPLB-pPIC9K质粒,10.0μl 10×4Buffer,4.0μl SacΙ,37℃酶切4hrs。用OmegaBio-Tek公司的E.Z.N.ATM的产物回收试剂盒将线性化片段回收,-20℃保存备用。
毕赤酵母感受态的制备:挑取毕赤酵母GS115单菌落,接种至含有30ml YPD培养基的250ml三角瓶中,28℃、200rpm培养过夜。取500ul的培养物接种至含有30ml新鲜YPD培养基的250ml三角摇瓶中,30℃、200rpm培养过夜,至OD600达到1-1.5。将培养液于4℃,1500g离心5min,用500ml冰预冷的无菌水将菌体沉淀重悬。4℃,1500g离心5min,用250ml冰预冷的无菌水将菌体沉淀重悬。4℃,1500g离心5min,用20ml冰预冷的1M山梨醇溶液将菌体沉淀重悬。4℃,1500g离心5min离心,用1ml冰预冷的1M山梨醇溶液将菌体沉淀重悬(每支感受态包含20OD细胞),分装至2ml离心管待用(100ul/支)。
取线性化的产物5μl分别与100μl新鲜毕赤酵母感受态菌体GS115混匀,移取至冰预冷的电转杯中,冰浴5min。以1.5kV、25μF、400Ω条件电击1次,立即加入1ml冰预冷的山梨醇溶液将菌体混匀,并移取菌液涂布于MD平板(20g/L葡萄糖,15-20g/L琼脂,900ml蒸馏水高压灭菌20min,冷却后超净台上加入100ml 10×YNB,2ml 500×Biotin,倒平板)上。置于28℃培养48-55h,至单菌落出现,得到ECSPLB-pPIC9K-GS115突变体库。
实施例3:磷脂酶突变体库的平板筛选
将ECSPLB-pPIC9K-GS115突变体所有菌点板到BMMY平板(1%酵母浸出物,2%蛋白胨,100mM磷酸钾PH6.0,1.34%YNB 4×10-5%生物素,0.5%甲醇,1%大豆卵磷脂,1.5%琼脂)上,30℃培养3天,结果如图1所示。图1中的四个平板,每个平板正中间一个菌为磷脂酶亲本,其它的菌为磷脂酶变体。挑选透明圈和浑浊圈比亲本圈大的磷脂酶正向变体。图1中,左上图中黑方块标记处为含磷脂酶变体154的菌,右上图中黑方块标记处为含磷脂酶变体2的菌,左下图中黑方块标记处为含磷脂酶变体10的菌,右下图中黑方块标记处为含磷脂酶变体17的菌。
实施例4:磷脂酶变体摇瓶发酵和磷脂酶活力检测
将在筛选平板上筛选到的磷脂酶变体2、10、17和154,挑取单克隆接种至30ml YPD液体培养基(培养GS115空宿主作为对照)。28℃,200rpm培养30-40hrs。取少量菌液接种至30ml BMGY(1%酵母浸出物,2%蛋白胨,100mM磷酸钾PH6.0,1.34%YNB 4×10-5%生物素,1%甘油)液体培养基(28℃、200rpm摇至OD600达到2-6。1500-3000g、4℃离心5min,收集细胞,去除上清,用30ml BMMY(1%酵母浸出物,2%蛋白胨,100mM磷酸钾pH6.0,1.34%YNB 4×10-5%生物素,0.5%甲醇)培养基等体积重悬细胞,28℃、200rpm条件下培养,使用0.5%甲醇进行诱导表达。每24小时,加甲醇至终浓度为0.5%以继续诱导,培养72小时后,收集上清液得到,用于目标磷脂酶的蛋白表达检测及酶活分析,以确定诱导后收集细胞的最佳时间。
结果如下表1所示。
表1:四个变体磷脂酶的酶活比较
磷脂酶变体2、10、17和154测序后,氨基酸突变位点汇总如表2所示。
表2:磷脂酶变体2、10、17和154氨基酸突变位点汇总
磷脂酶变体 | 突变数 | 具体突变位置 |
磷脂酶变体2 | 1个 | I342N |
磷脂酶变体10 | 3个 | I182N、E323G、L339F |
磷脂酶变体17 | 3个 | D274E、L339I、A500T |
磷脂酶变体154 | 2个 | Q221K、R367W |
实施例5:亲本磷脂酶和变体磷脂酶的温度稳定性分析
分别将亲本磷脂酶和变体磷脂酶酶液按照500μl量分装成若干小份,放置于4℃、25℃、37℃、40℃、45℃、50℃、55℃、60℃和65℃的水浴锅中保温1小时,然后进行磷脂酶活力的检测,以未孵育的样品的磷脂酶活力值为100%,其它温度点的磷脂酶酶活除以该酶活力值从而得到其相对酶活值,以相对酶活为纵坐标,温度为横坐标,以平滑曲线连接各温度点的相对酶活,结果见附图2。
图2的结果显示本发明中变体磷脂酶17在50℃-55℃(脱胶温度范围)的热稳定性好于亲本磷脂酶,变体磷脂酶154在50℃-55℃(脱胶温度范围)的热稳定性与亲本磷脂酶相当,变体磷脂酶2在50℃-55℃(脱胶温度范围)的热稳定性不如亲本磷脂酶。
实施例6、亲本磷脂酶和变体磷脂酶2大豆毛油脱胶效果
称200g大豆毛油(上海嘉里粮油工业有限公司)至烧杯中,高速分散均质机均质1分钟,然后边搅拌边加热大豆毛油至55℃,加入水和变体磷脂酶2(共6g),均质1分钟,50℃150-160rpm搅拌水解脱胶4小时,加热至85℃10分钟灭酶,离心12000rpm离心10分钟,取上层油测定无机磷含量。
磷脂酶 | 无机磷含量(ppm) |
变体磷脂酶2 | 55.3 |
亲本磷脂酶 | 80.9 |
大豆毛油 | 514.5 |
实施例7、亲本磷脂酶和变体磷脂酶2的最适温度曲线
分别在30℃、32℃、35℃、40℃、45℃、50℃、53℃和55℃一系列的温度下测定变体磷脂酶2和亲本磷脂酶的活力。以最高磷脂酶酶活的温度点的磷脂酶酶活作为100%,其它温度点的磷脂酶酶活除以该最高酶活,从而得到该温度点的相对磷脂酶酶活,以相对磷脂酶酶活为纵坐标,温度点为横坐标,以平滑曲线依次连接各温度点的相对酶活,将这些相对酶活以平滑曲线连接,结果见图3和图4。
图3和图4的结果显示,亲本磷脂酶的最适合作用温度为40℃,变体磷脂酶2的最适作用温度为45℃。
实施例8、亲本磷脂酶和变体磷脂酶2的最适pH值曲线
配制0.2M的pH值分别为3.0、3.5、4.0、4.5、5.5、6.1和6.5的乙酸-乙酸钠缓冲液,分别在以上不同pH条件下的检测本发明亲本磷脂酶和变体磷脂酶2的磷脂酶活力值。以最高磷脂酶活力的pH点的磷脂酶活作为100%,其它pH的磷脂酶酶活除以该最高酶活,从而得到该pH的相对磷脂酶酶活,以相对磷脂酶酶活为纵坐标,pH值为横坐标,以平滑曲线依次连接各pH的相对磷脂酶酶活。本发明多肽在不同pH值条件下的相对磷脂酶酶活结果见图5和图6。
由图5和图6可以看出,亲本磷脂酶在pH为4时的磷脂酶活力最高,变体磷脂酶2在pH4.5时的磷脂酶活力最高。
实施例9、金属离子及EDTA对亲本磷脂酶和变体磷脂酶2的磷脂酶活力的影响
分别配制250mM的钙离子、镁离子、锰离子、锌离子、钴离子、镍离子、铁离子、钠离子、钾离子母液,配制250mM的乙二胺四乙酸(EDTA)母液。将上述母液分别按照5.0mM的量加入至磷脂酶的反应体系中,测定酶活。以不添加任何离子及EDTA的样品所测的磷脂酶酶活力值为100%,作为对照,计算其它添加物的样品磷脂酶及脂肪酶的相对活力值,结果见图7和图8。
由图7和图8可以看出,5mM的锰离子和钴离子对亲本磷脂酶有略微的激活作用,其它金属离子对亲本磷脂酶无激活作用;而检测的几种金属离子对变体磷脂酶2无激活作用。
序列表
<110> 丰益(上海)生物技术研发中心有限公司
<120> 磷脂酶及其应用
<130> 167236
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 1638
<212> DNA
<213> 人工序列
<220>
<223> 编码亲本磷脂酶多肽的多核苷酸序列
<400> 1
gcccccgccc caggtgcagt gcctgtgcct cctttgtacg ctgagggtgt cctcgagctt 60
gcgaagaggc aaagcggtct gacccggtac cagccggtgg aggtgccctg ccctgacact 120
gctctcgtga gagatgcaac gggattgggc actccagaag ccgagtacat cgcctcccga 180
aaagaaaagg cagatgccgc gcttgcgcag tggctagaga ggcaaggctc gttcggcgac 240
tcgagtcagc ctaccgttgc tttggcatct agtggtggag gctaccgtgc acttctcgcc 300
ggtgctggtg tcatcaaggc attcgacgct cgcgagggta gcaacgaggt cagcggcctc 360
tatcaagcat tgacttacca atccggtctt tccggcggcg catggctcct ttcttcccag 420
tcaggcaatg actggcctac tatcagctat ttgcaagaga acctctggga ggatgctttc 480
gaggtctctc ttctgcttcc ggcgaacctt ctctccttct ccggtcttac ccagtatggt 540
gcaatcacca cagagattct ctccaaggaa ggtgccggct ttgacacgac catcatcgac 600
ccgtggggcc gtcttctctc ctaccaactc ctcgagggcg cattcggcgg cgtagacacg 660
caaatgtcca ccctttcatc cctcagcaac ttcaccgacc acaacgttcc cttccctatc 720
atcacggcac tcggcgtcgt ggccagggag aaccaatgca cccctcccga cgacgccacc 780
cagtacgaat tccaccccta cgagttcggc tcctgggacg acggcctctc cgccttcgcc 840
aacgtcgaat tcctcggctc caacctcaac aatggccagc ccgaagacgc cagccagtgt 900
gtcgccgagt acgacaacct cggctacgtc ctcggcacct cctccaacat cttcccagcc 960
atttgcgaac ccctctcacc cgccaacagc accgacgacc ccctcgccaa cgtcctcgaa 1020
ggcatcatca gcctcgccca cgagcccgtc ttcgaagacc tcttcgccct ctaccgcaac 1080
cccttcttca acctctcgcg gtccagccgc gtctcgcaag acgaagaact cctcctcctc 1140
gacggcggct tcagcaacca gaacgtcccc atctggccct tcatcgtcga agcccgcgac 1200
gtcgacgtcc tgatcgtcaa cgacaactcc gccgacaccg aggacaacta cccgaacggc 1260
acggcgccgc accagacgta cttgcaggcg cagctgaacg gcttggccaa gatgccgttc 1320
gtgcctgatg tgaacacgtt cgtctcgcag ggcctgaacc agcgtgcgac gttcttcggc 1380
tgcgatgaac cggacacgac gttcattgtc tacctgccaa acagggagta cagttttgcg 1440
tcgggccagt cgacgggcaa ggtcgtgtac tccaaagacg agacgagggc tatgattgcc 1500
aacggcgtgc aggttggtac gcaggacggc gaggagggtt ggggcctgtg cttggcttgt 1560
gcgattaaga gtcgcgagga gggtttgcct gagggctgtg cggcttgctt cgataagcac 1620
tgctatcgtc agagctga 1638
<210> 2
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 亲本磷脂酶多肽的氨基酸序列
<400> 2
Ala Pro Ala Pro Gly Ala Val Pro Val Pro Pro Leu Tyr Ala Glu Gly
1 5 10 15
Val Leu Glu Leu Ala Lys Arg Gln Ser Gly Leu Thr Arg Tyr Gln Pro
20 25 30
Val Glu Val Pro Cys Pro Asp Thr Ala Leu Val Arg Asp Ala Thr Gly
35 40 45
Leu Gly Thr Pro Glu Ala Glu Tyr Ile Ala Ser Arg Lys Glu Lys Ala
50 55 60
Asp Ala Ala Leu Ala Gln Trp Leu Glu Arg Gln Gly Ser Phe Gly Asp
65 70 75 80
Ser Ser Gln Pro Thr Val Ala Leu Ala Ser Ser Gly Gly Gly Tyr Arg
85 90 95
Ala Leu Leu Ala Gly Ala Gly Val Ile Lys Ala Phe Asp Ala Arg Glu
100 105 110
Gly Ser Asn Glu Val Ser Gly Leu Tyr Gln Ala Leu Thr Tyr Gln Ser
115 120 125
Gly Leu Ser Gly Gly Ala Trp Leu Leu Ser Ser Gln Ser Gly Asn Asp
130 135 140
Trp Pro Thr Ile Ser Tyr Leu Gln Glu Asn Leu Trp Glu Asp Ala Phe
145 150 155 160
Glu Val Ser Leu Leu Leu Pro Ala Asn Leu Leu Ser Phe Ser Gly Leu
165 170 175
Thr Gln Tyr Gly Ala Ile Thr Thr Glu Ile Leu Ser Lys Glu Gly Ala
180 185 190
Gly Phe Asp Thr Thr Ile Ile Asp Pro Trp Gly Arg Leu Leu Ser Tyr
195 200 205
Gln Leu Leu Glu Gly Ala Phe Gly Gly Val Asp Thr Gln Met Ser Thr
210 215 220
Leu Ser Ser Leu Ser Asn Phe Thr Asp His Asn Val Pro Phe Pro Ile
225 230 235 240
Ile Thr Ala Leu Gly Val Val Ala Arg Glu Asn Gln Cys Thr Pro Pro
245 250 255
Asp Asp Ala Thr Gln Tyr Glu Phe His Pro Tyr Glu Phe Gly Ser Trp
260 265 270
Asp Asp Gly Leu Ser Ala Phe Ala Asn Val Glu Phe Leu Gly Ser Asn
275 280 285
Leu Asn Asn Gly Gln Pro Glu Asp Ala Ser Gln Cys Val Ala Glu Tyr
290 295 300
Asp Asn Leu Gly Tyr Val Leu Gly Thr Ser Ser Asn Ile Phe Pro Ala
305 310 315 320
Ile Cys Glu Pro Leu Ser Pro Ala Asn Ser Thr Asp Asp Pro Leu Ala
325 330 335
Asn Val Leu Glu Gly Ile Ile Ser Leu Ala His Glu Pro Val Phe Glu
340 345 350
Asp Leu Phe Ala Leu Tyr Arg Asn Pro Phe Phe Asn Leu Ser Arg Ser
355 360 365
Ser Arg Val Ser Gln Asp Glu Glu Leu Leu Leu Leu Asp Gly Gly Phe
370 375 380
Ser Asn Gln Asn Val Pro Ile Trp Pro Phe Ile Val Glu Ala Arg Asp
385 390 395 400
Val Asp Val Leu Ile Val Asn Asp Asn Ser Ala Asp Thr Glu Asp Asn
405 410 415
Tyr Pro Asn Gly Thr Ala Pro His Gln Thr Tyr Leu Gln Ala Gln Leu
420 425 430
Asn Gly Leu Ala Lys Met Pro Phe Val Pro Asp Val Asn Thr Phe Val
435 440 445
Ser Gln Gly Leu Asn Gln Arg Ala Thr Phe Phe Gly Cys Asp Glu Pro
450 455 460
Asp Thr Thr Phe Ile Val Tyr Leu Pro Asn Arg Glu Tyr Ser Phe Ala
465 470 475 480
Ser Gly Gln Ser Thr Gly Lys Val Val Tyr Ser Lys Asp Glu Thr Arg
485 490 495
Ala Met Ile Ala Asn Gly Val Gln Val Gly Thr Gln Asp Gly Glu Glu
500 505 510
Gly Trp Gly Leu Cys Leu Ala Cys Ala Ile Lys Ser Arg Glu Glu Gly
515 520 525
Leu Pro Glu Gly Cys Ala Ala Cys Phe Asp Lys His Cys Tyr Arg Gln
530 535 540
Ser
545
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 3
aaacctaggg cccccgcccc aggtg 25
<210> 4
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 4
aaagcggccg ctcagctctg acgatagc 28
<210> 5
<211> 1638
<212> DNA
<213> 人工序列
<220>
<223> 磷脂酶变体2的多核苷酸序列
<400> 5
gcccccgccc caggtgcagt gcctgtgcct cctttgtacg ctgagggtgt cctcgagctt 60
gcgaagaggc aaagcggtct gacccggtac cagccggtgg aggtgccctg ccctgacact 120
gctctcgtga gagatgcaac gggattgggc actccagaag ccgagtacat cgcctcccga 180
aaagaaaagg cagatgccgc gcttgcgcag tggctagaga ggcaaggctc gttcggcgac 240
tcgagtcagc ctaccgttgc tttggcatct agtggtggag gctaccgtgc acttctcgcc 300
ggtgctggtg tcatcaaggc attcgacgct cgcgagggta gcaacgaggt cagcggcctc 360
tatcaagcat tgacttacca atccggtctt tccggcggcg catggctcct ttcttcccag 420
tcaggcaatg actggcctac tatcagctat ttgcaagaga acctctggga ggatgctttc 480
gaggtctctc ttctgcttcc ggcgaacctt ctctccttct ccggtcttac ccagtatggt 540
gcaatcacca cagagattct ctccaaggaa ggtgccggct ttgacacgac catcatcgac 600
ccgtggggcc gtcttctctc ctaccaactc ctcgagggcg cattcggcgg cgtagacacg 660
caaatgtcca ccctttcatc cctcagcaac ttcaccgacc acaacgttcc cttccctatc 720
atcacggcac tcggcgtcgt ggccagggag aaccaatgca cccctcccga cgacgccacc 780
cagtacgaat tccaccccta cgagttcggc tcctgggacg acggcctctc cgccttcgcc 840
aacgtcgaat tcctcggctc caacctcaac aatggccagc ccgaagacgc cagccagtgt 900
gtcgccgagt acgacaacct cggctacgtc ctcggcacct cctccaacat cttcccagcc 960
atttgcgaac ccctctcacc cgccaacagc accgacgacc ccctcgccaa cgtcctcgaa 1020
ggcaacatca gcctcgccca cgagcccgtc ttcgaagacc tcttcgccct ctaccgcaac 1080
cccttcttca acctctcgcg gtccagccgc gtctcgcaag acgaagaact cctcctcctc 1140
gacggcggct tcagcaacca gaacgtcccc atctggccct tcatcgtcga agcccgcgac 1200
gtcgacgtcc tgatcgtcaa cgacaactcc gccgacaccg aggacaacta cccgaacggc 1260
acggcgccgc accagacgta cttgcaggcg cagctgaacg gcttggccaa gatgccgttc 1320
gtgcctgatg tgaacacgtt cgtctcgcag ggcctgaacc agcgtgcgac gttcttcggc 1380
tgcgatgaac cggacacgac gttcattgtc tacctgccaa acagggagta cagttttgcg 1440
tcgggccagt cgacgggcaa ggtcgtgtac tccaaagacg agacgagggc tatgattgcc 1500
aacggcgtgc aggttggtac gcaggacggc gaggagggtt ggggcctgtg cttggcttgt 1560
gcgattaaga gtcgcgagga gggtttgcct gagggctgtg cggcttgctt cgataagcac 1620
tgctatcgtc agagctga 1638
<210> 6
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 磷脂酶变体2的多肽的氨基酸序列
<400> 6
Ala Pro Ala Pro Gly Ala Val Pro Val Pro Pro Leu Tyr Ala Glu Gly
1 5 10 15
Val Leu Glu Leu Ala Lys Arg Gln Ser Gly Leu Thr Arg Tyr Gln Pro
20 25 30
Val Glu Val Pro Cys Pro Asp Thr Ala Leu Val Arg Asp Ala Thr Gly
35 40 45
Leu Gly Thr Pro Glu Ala Glu Tyr Ile Ala Ser Arg Lys Glu Lys Ala
50 55 60
Asp Ala Ala Leu Ala Gln Trp Leu Glu Arg Gln Gly Ser Phe Gly Asp
65 70 75 80
Ser Ser Gln Pro Thr Val Ala Leu Ala Ser Ser Gly Gly Gly Tyr Arg
85 90 95
Ala Leu Leu Ala Gly Ala Gly Val Ile Lys Ala Phe Asp Ala Arg Glu
100 105 110
Gly Ser Asn Glu Val Ser Gly Leu Tyr Gln Ala Leu Thr Tyr Gln Ser
115 120 125
Gly Leu Ser Gly Gly Ala Trp Leu Leu Ser Ser Gln Ser Gly Asn Asp
130 135 140
Trp Pro Thr Ile Ser Tyr Leu Gln Glu Asn Leu Trp Glu Asp Ala Phe
145 150 155 160
Glu Val Ser Leu Leu Leu Pro Ala Asn Leu Leu Ser Phe Ser Gly Leu
165 170 175
Thr Gln Tyr Gly Ala Ile Thr Thr Glu Ile Leu Ser Lys Glu Gly Ala
180 185 190
Gly Phe Asp Thr Thr Ile Ile Asp Pro Trp Gly Arg Leu Leu Ser Tyr
195 200 205
Gln Leu Leu Glu Gly Ala Phe Gly Gly Val Asp Thr Gln Met Ser Thr
210 215 220
Leu Ser Ser Leu Ser Asn Phe Thr Asp His Asn Val Pro Phe Pro Ile
225 230 235 240
Ile Thr Ala Leu Gly Val Val Ala Arg Glu Asn Gln Cys Thr Pro Pro
245 250 255
Asp Asp Ala Thr Gln Tyr Glu Phe His Pro Tyr Glu Phe Gly Ser Trp
260 265 270
Asp Asp Gly Leu Ser Ala Phe Ala Asn Val Glu Phe Leu Gly Ser Asn
275 280 285
Leu Asn Asn Gly Gln Pro Glu Asp Ala Ser Gln Cys Val Ala Glu Tyr
290 295 300
Asp Asn Leu Gly Tyr Val Leu Gly Thr Ser Ser Asn Ile Phe Pro Ala
305 310 315 320
Ile Cys Glu Pro Leu Ser Pro Ala Asn Ser Thr Asp Asp Pro Leu Ala
325 330 335
Asn Val Leu Glu Gly Asn Ile Ser Leu Ala His Glu Pro Val Phe Glu
340 345 350
Asp Leu Phe Ala Leu Tyr Arg Asn Pro Phe Phe Asn Leu Ser Arg Ser
355 360 365
Ser Arg Val Ser Gln Asp Glu Glu Leu Leu Leu Leu Asp Gly Gly Phe
370 375 380
Ser Asn Gln Asn Val Pro Ile Trp Pro Phe Ile Val Glu Ala Arg Asp
385 390 395 400
Val Asp Val Leu Ile Val Asn Asp Asn Ser Ala Asp Thr Glu Asp Asn
405 410 415
Tyr Pro Asn Gly Thr Ala Pro His Gln Thr Tyr Leu Gln Ala Gln Leu
420 425 430
Asn Gly Leu Ala Lys Met Pro Phe Val Pro Asp Val Asn Thr Phe Val
435 440 445
Ser Gln Gly Leu Asn Gln Arg Ala Thr Phe Phe Gly Cys Asp Glu Pro
450 455 460
Asp Thr Thr Phe Ile Val Tyr Leu Pro Asn Arg Glu Tyr Ser Phe Ala
465 470 475 480
Ser Gly Gln Ser Thr Gly Lys Val Val Tyr Ser Lys Asp Glu Thr Arg
485 490 495
Ala Met Ile Ala Asn Gly Val Gln Val Gly Thr Gln Asp Gly Glu Glu
500 505 510
Gly Trp Gly Leu Cys Leu Ala Cys Ala Ile Lys Ser Arg Glu Glu Gly
515 520 525
Leu Pro Glu Gly Cys Ala Ala Cys Phe Asp Lys His Cys Tyr Arg Gln
530 535 540
Ser
545
<210> 7
<211> 1638
<212> DNA
<213> 人工序列
<220>
<223> 磷脂酶变体10的多核苷酸序列
<400> 7
gcccccgccc caggtgcagt gcctgtgcct cctttgtacg ctgagggtgt cctcgagctt 60
gcgaagaggc aaagcggtct gacccggtac cagccggtgg aggtgccctg ccctgacact 120
gctctcgtga gagatgcaac gggattgggc actccagaag ccgagtacat cgcctcccga 180
aaagaaaagg cagatgccgc gcttgcgcag tggctagaga ggcaaggctc gttcggcgac 240
tcgagtcagc ctaccgttgc tttggcatct agtggcggag gctaccgtgc acttctcgcc 300
ggtgctggtg tcatcaaggc attcgacgct cgcgagggta gcaacgaggt cagcggcctc 360
tatcaagcat tgacttacca atccggtctt tccggcggcg catggctcct ttcttcccag 420
tcaggcaatg actggcctac tatcagctat ttgcaagaga acctctggga ggatgctttc 480
gaggtctctc ttctgcttcc ggcgaacctt ctctccttct ccggtcttac ccagtatggt 540
gcagtcacca cagagattct ctccaaggaa ggtgccggct ttgacacgac catcatcgac 600
ccgtggggcc gtcttctctc ctaccaactc ctcgagggcg cattcggcgg cgtagacacg 660
caaatgtcca ccctttcatc cctcagcaac ttcaccgacc acaacgttcc cttccctatc 720
atcacggcac tcggcgtcgt ggccagggag aaccaatgca cccctcccga cgacgccacc 780
cagtacgaat tccaccccta cgagttcggc tcctgggacg acggcctctc cgccttcgcc 840
aacgtcgaat tcctcggctc caacctcaac aatggccagc ccgaagacgc cagccagtgt 900
gtcgccgagt acgacaacct cggctacgtc ctcggcacct cctccaacat cttcccagcc 960
atttgcggac ccctctcacc cgccaacagc accgacgacc ccctcgccaa cgtcttcgaa 1020
ggcatcatca gcctcgccca cgagcccgtc ttcgaagacc tcttcgccct ctaccgcaac 1080
cccttcttca acctctcgcg gtccagccgt gtctcgcaag acgaagaact cctcctcctc 1140
gacggcggct tcagcaacca gaacgtcccc atctggccct tcatcgtcga agcccgcgac 1200
gtcgacgtcc tgatcgtcaa cgacaactcc gccgacaccg aggacaacta cccgaacggc 1260
acggcgccgc accagacgta cttgcaggcg cagctgaacg gcttggccaa gatgccgttc 1320
gtgcctgatg tgaacacgtt cgtctcgcag ggcctgaacc agcgtgcgac gttcttcggc 1380
tgtgatgaac cggacacgac gttcattgtc tacctgccaa acagggagta cagttttgcg 1440
tcgggccagt cgacgggcaa ggtcgtgtac tccaaagacg agacgagggc tatgattgcc 1500
aacggcgtgc aggttggtac gcaggacggc gaggagggtt ggggcctgtg cttggcttgt 1560
gcgattaaga gtcgcgagga gggtttgcct gagggctgtg cggcttgctt cgataagcac 1620
tgctatcgtc agagctga 1638
<210> 8
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 磷脂酶变体10的多肽的氨基酸序列
<400> 8
Ala Pro Ala Pro Gly Ala Val Pro Val Pro Pro Leu Tyr Ala Glu Gly
1 5 10 15
Val Leu Glu Leu Ala Lys Arg Gln Ser Gly Leu Thr Arg Tyr Gln Pro
20 25 30
Val Glu Val Pro Cys Pro Asp Thr Ala Leu Val Arg Asp Ala Thr Gly
35 40 45
Leu Gly Thr Pro Glu Ala Glu Tyr Ile Ala Ser Arg Lys Glu Lys Ala
50 55 60
Asp Ala Ala Leu Ala Gln Trp Leu Glu Arg Gln Gly Ser Phe Gly Asp
65 70 75 80
Ser Ser Gln Pro Thr Val Ala Leu Ala Ser Ser Gly Gly Gly Tyr Arg
85 90 95
Ala Leu Leu Ala Gly Ala Gly Val Ile Lys Ala Phe Asp Ala Arg Glu
100 105 110
Gly Ser Asn Glu Val Ser Gly Leu Tyr Gln Ala Leu Thr Tyr Gln Ser
115 120 125
Gly Leu Ser Gly Gly Ala Trp Leu Leu Ser Ser Gln Ser Gly Asn Asp
130 135 140
Trp Pro Thr Ile Ser Tyr Leu Gln Glu Asn Leu Trp Glu Asp Ala Phe
145 150 155 160
Glu Val Ser Leu Leu Leu Pro Ala Asn Leu Leu Ser Phe Ser Gly Leu
165 170 175
Thr Gln Tyr Gly Ala Val Thr Thr Glu Ile Leu Ser Lys Glu Gly Ala
180 185 190
Gly Phe Asp Thr Thr Ile Ile Asp Pro Trp Gly Arg Leu Leu Ser Tyr
195 200 205
Gln Leu Leu Glu Gly Ala Phe Gly Gly Val Asp Thr Gln Met Ser Thr
210 215 220
Leu Ser Ser Leu Ser Asn Phe Thr Asp His Asn Val Pro Phe Pro Ile
225 230 235 240
Ile Thr Ala Leu Gly Val Val Ala Arg Glu Asn Gln Cys Thr Pro Pro
245 250 255
Asp Asp Ala Thr Gln Tyr Glu Phe His Pro Tyr Glu Phe Gly Ser Trp
260 265 270
Asp Asp Gly Leu Ser Ala Phe Ala Asn Val Glu Phe Leu Gly Ser Asn
275 280 285
Leu Asn Asn Gly Gln Pro Glu Asp Ala Ser Gln Cys Val Ala Glu Tyr
290 295 300
Asp Asn Leu Gly Tyr Val Leu Gly Thr Ser Ser Asn Ile Phe Pro Ala
305 310 315 320
Ile Cys Gly Pro Leu Ser Pro Ala Asn Ser Thr Asp Asp Pro Leu Ala
325 330 335
Asn Val Phe Glu Gly Ile Ile Ser Leu Ala His Glu Pro Val Phe Glu
340 345 350
Asp Leu Phe Ala Leu Tyr Arg Asn Pro Phe Phe Asn Leu Ser Arg Ser
355 360 365
Ser Arg Val Ser Gln Asp Glu Glu Leu Leu Leu Leu Asp Gly Gly Phe
370 375 380
Ser Asn Gln Asn Val Pro Ile Trp Pro Phe Ile Val Glu Ala Arg Asp
385 390 395 400
Val Asp Val Leu Ile Val Asn Asp Asn Ser Ala Asp Thr Glu Asp Asn
405 410 415
Tyr Pro Asn Gly Thr Ala Pro His Gln Thr Tyr Leu Gln Ala Gln Leu
420 425 430
Asn Gly Leu Ala Lys Met Pro Phe Val Pro Asp Val Asn Thr Phe Val
435 440 445
Ser Gln Gly Leu Asn Gln Arg Ala Thr Phe Phe Gly Cys Asp Glu Pro
450 455 460
Asp Thr Thr Phe Ile Val Tyr Leu Pro Asn Arg Glu Tyr Ser Phe Ala
465 470 475 480
Ser Gly Gln Ser Thr Gly Lys Val Val Tyr Ser Lys Asp Glu Thr Arg
485 490 495
Ala Met Ile Ala Asn Gly Val Gln Val Gly Thr Gln Asp Gly Glu Glu
500 505 510
Gly Trp Gly Leu Cys Leu Ala Cys Ala Ile Lys Ser Arg Glu Glu Gly
515 520 525
Leu Pro Glu Gly Cys Ala Ala Cys Phe Asp Lys His Cys Tyr Arg Gln
530 535 540
Ser
545
<210> 9
<211> 1638
<212> DNA
<213> 人工序列
<220>
<223> 磷脂酶变体17的多核苷酸序列
<400> 9
gcccccgccc caggtgcagt gcctgtgcct cctttgtacg ctgagggtgt cctcgagctt 60
gcgaagaggc aaagcggtct gacccggtac cagccggtgg aggtgccctg ccctgacact 120
gctctcgtga gagatgcaac gggattgggc actccagaag ccgagtacat cgcctcccga 180
aaagaaaagg cagatgccgc gcttgcgcag tggctagaga ggcaaggctc gttcggcgac 240
tcgagtcagc ctaccgttgc tttggcatct agtggtggag gctaccgtgc acttctcgcc 300
ggtgctggtg tcatcaaggc attcgacgct cgcgagggta gcaacgaggt cagcggcctc 360
tatcaagcat tgacttacca atccggtctt tccggcggcg catggctcct ttcttcccag 420
tcaggcaatg actggcctac tatcagctat ttgcaagaga acctctggga ggatgctttc 480
gaggtctctc ttctgcttcc ggcgaacctt ctctccttct ccggtcttac ccagtatggt 540
gcaatcacca cagagattct ctccaaggaa ggtgccggct ttgacacgac catcatcgac 600
ccgtggggcc gtcttctctc ctaccaactc ctcgagggcg cattcggcgg cgtagacacg 660
caaatgtcca ccctttcatc cctcagcaac ttcaccgacc acaacgttcc cttccctatc 720
atcacggcac tcggcgtcgt ggccagggag aaccaatgca cccctcccga cgacgccacc 780
cagtacgaat tccaccccta cgagttcggc tcctgggacg aaggcctctc cgccttcgcc 840
aacgtcgaat tcctcggctc caacctcaac aatggccagc ccgaagacgc cagccagtgt 900
gtcgccgagt acgacaacct cggctacgtc ctcggcacct cctccaacat cttcccagcc 960
atttgcgaac ccctctcacc cgccaacagc accgacgacc ccctcgccaa cgtcatcgaa 1020
ggcatcatca gcctcgccca cgagcccgtc ttcgaagacc tcttcgccct ctaccgcaac 1080
cccttcttca acctctcgcg gtccagccgc gtctcgcaag acgaagaact cctcctcctc 1140
gacggcggct tcagcaacca gaacgtcccc atctggccct tcatcgtcga agcccgcgac 1200
gtcgacgtcc tgatcgtcaa cgacaactcc gccgacaccg aggacaacta cccgaacggc 1260
acggcgccgc accagacgta cttgcaggcg cagctgaacg gcttggccaa gatgccgttc 1320
gtgcctgatg tgaacacgtt cgtctcgcag ggcctgaacc agcgtgcgac gttcttcggc 1380
tgcgatgaac cggacacgac gttcattgtc tacctgccaa acagggagta cagttttgcg 1440
tcgggccagt cgacgggcaa ggtcgtgtac tccaaagacg agacgagggc tatgattacc 1500
aacggcgtgc aggttggtac gcaggacggc gaggagggtt ggggcctgtg cttggcttgt 1560
gcgattaaga gtcgcgagga gggtttgcct gagggctgtg cggcttgctt cgataagcac 1620
tgctatcgtc agagctga 1638
<210> 10
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 磷脂酶变体17的多肽的氨基酸序列
<400> 10
Ala Pro Ala Pro Gly Ala Val Pro Val Pro Pro Leu Tyr Ala Glu Gly
1 5 10 15
Val Leu Glu Leu Ala Lys Arg Gln Ser Gly Leu Thr Arg Tyr Gln Pro
20 25 30
Val Glu Val Pro Cys Pro Asp Thr Ala Leu Val Arg Asp Ala Thr Gly
35 40 45
Leu Gly Thr Pro Glu Ala Glu Tyr Ile Ala Ser Arg Lys Glu Lys Ala
50 55 60
Asp Ala Ala Leu Ala Gln Trp Leu Glu Arg Gln Gly Ser Phe Gly Asp
65 70 75 80
Ser Ser Gln Pro Thr Val Ala Leu Ala Ser Ser Gly Gly Gly Tyr Arg
85 90 95
Ala Leu Leu Ala Gly Ala Gly Val Ile Lys Ala Phe Asp Ala Arg Glu
100 105 110
Gly Ser Asn Glu Val Ser Gly Leu Tyr Gln Ala Leu Thr Tyr Gln Ser
115 120 125
Gly Leu Ser Gly Gly Ala Trp Leu Leu Ser Ser Gln Ser Gly Asn Asp
130 135 140
Trp Pro Thr Ile Ser Tyr Leu Gln Glu Asn Leu Trp Glu Asp Ala Phe
145 150 155 160
Glu Val Ser Leu Leu Leu Pro Ala Asn Leu Leu Ser Phe Ser Gly Leu
165 170 175
Thr Gln Tyr Gly Ala Ile Thr Thr Glu Ile Leu Ser Lys Glu Gly Ala
180 185 190
Gly Phe Asp Thr Thr Ile Ile Asp Pro Trp Gly Arg Leu Leu Ser Tyr
195 200 205
Gln Leu Leu Glu Gly Ala Phe Gly Gly Val Asp Thr Gln Met Ser Thr
210 215 220
Leu Ser Ser Leu Ser Asn Phe Thr Asp His Asn Val Pro Phe Pro Ile
225 230 235 240
Ile Thr Ala Leu Gly Val Val Ala Arg Glu Asn Gln Cys Thr Pro Pro
245 250 255
Asp Asp Ala Thr Gln Tyr Glu Phe His Pro Tyr Glu Phe Gly Ser Trp
260 265 270
Asp Glu Gly Leu Ser Ala Phe Ala Asn Val Glu Phe Leu Gly Ser Asn
275 280 285
Leu Asn Asn Gly Gln Pro Glu Asp Ala Ser Gln Cys Val Ala Glu Tyr
290 295 300
Asp Asn Leu Gly Tyr Val Leu Gly Thr Ser Ser Asn Ile Phe Pro Ala
305 310 315 320
Ile Cys Glu Pro Leu Ser Pro Ala Asn Ser Thr Asp Asp Pro Leu Ala
325 330 335
Asn Val Ile Glu Gly Ile Ile Ser Leu Ala His Glu Pro Val Phe Glu
340 345 350
Asp Leu Phe Ala Leu Tyr Arg Asn Pro Phe Phe Asn Leu Ser Arg Ser
355 360 365
Ser Arg Val Ser Gln Asp Glu Glu Leu Leu Leu Leu Asp Gly Gly Phe
370 375 380
Ser Asn Gln Asn Val Pro Ile Trp Pro Phe Ile Val Glu Ala Arg Asp
385 390 395 400
Val Asp Val Leu Ile Val Asn Asp Asn Ser Ala Asp Thr Glu Asp Asn
405 410 415
Tyr Pro Asn Gly Thr Ala Pro His Gln Thr Tyr Leu Gln Ala Gln Leu
420 425 430
Asn Gly Leu Ala Lys Met Pro Phe Val Pro Asp Val Asn Thr Phe Val
435 440 445
Ser Gln Gly Leu Asn Gln Arg Ala Thr Phe Phe Gly Cys Asp Glu Pro
450 455 460
Asp Thr Thr Phe Ile Val Tyr Leu Pro Asn Arg Glu Tyr Ser Phe Ala
465 470 475 480
Ser Gly Gln Ser Thr Gly Lys Val Val Tyr Ser Lys Asp Glu Thr Arg
485 490 495
Ala Met Ile Thr Asn Gly Val Gln Val Gly Thr Gln Asp Gly Glu Glu
500 505 510
Gly Trp Gly Leu Cys Leu Ala Cys Ala Ile Lys Ser Arg Glu Glu Gly
515 520 525
Leu Pro Glu Gly Cys Ala Ala Cys Phe Asp Lys His Cys Tyr Arg Gln
530 535 540
Ser
545
<210> 11
<211> 1638
<212> DNA
<213> 人工序列
<220>
<223> 磷脂酶变体154的多核苷酸序列
<400> 11
gcccccgccc caggtgcagt gcctgtgcct cctttgtacg ctgagggtgt cctcgagctt 60
gcgaagaggc aaagcggtct gacccggtac cagccggtgg aggtgccctg ccctgacact 120
gctctcgtga gagatgcaac gggattgggc actccagaag ccgagtacat cgcctcccga 180
aaagaaaagg cagatgccgc gcttgcgcag tggctagaga ggcaaggctc gttcggcgac 240
tcgagtcagc ctaccgttgc tttggcatct agtggtggag gctaccgtgc acttctcgcc 300
ggtgctggtg tcatcaaggc attcgacgct cgcgagggta gcaacgaggt gagcggcctc 360
tatcaagcat tgacttacca atccggtctt tccggcggcg catggctcct ttcttcccag 420
tcaggcaatg actggcctac tatcagctat ttgcaagaga acctctggga ggatgctttc 480
gaggtctctc ttctgcttcc ggcgaacctt ctctccttct ccggtcttac ccagtatggt 540
gcaatcacca cagagattct ctccaaggaa ggtgccggct ttgacacgac catcatcgac 600
ccgtggggcc gtcttctctc ctaccaactc ctcgagggcg cattcggcgg cgtagacacg 660
aaaatgtcca ccctttcatc cctcagcaac ttcaccgacc acaacgttcc cttccctatc 720
atcacggcac tcggcgtcgt ggccagggag aaccaatgca cccctcccga cgacgccacc 780
cagtacgaat tccaccccta cgagttcggc tcctgggacg acggcctctc cgccttcgcc 840
aacgtcgaat tcctcggctc caacctcaac aatggccagc ccgaagacgc cagccagtgt 900
gtcgccgagt acgacaacct cggctacgtc ctcggcacct cctccaacat cttcccagcc 960
atttgcgaac ccctctcacc cgccaacagc accgacgacc ccctcgccaa cgtcctcgaa 1020
ggcatcatca gcctcgccca cgagcccgtc ttcgaagacc tcttcgccct ctaccgcaac 1080
cccttcttca acctctcgtg gtccagccgc gtctcgcaag acgaagaact cctcctcctc 1140
gacggcggct tcagcaacca gaacgtcccc atctggccct tcatcgtcga agcccgcgac 1200
gtcgacgtcc tgatcgtcaa cgacaactcc gccgacaccg aggacaacta cccgaacggc 1260
acggcgccgc accagacgta cttgcaggcg cagctgaacg gcttggccaa gatgccgttc 1320
gtgcctgatg tgaacacgtt cgtctcgcag ggcctgaacc agcgtgcgac gttcttcggc 1380
tgcgatgaac cggacacgac gttcattgtc tacctgccaa acagggagta cagttttgcg 1440
tcgggccagt cgacgggcaa ggtcgtgtac tccaaagacg agacgagggc tatgattgcc 1500
aacggcgtgc aggttggtac gcaggacggc gaggagggtt ggggcctgtg cttggcttgt 1560
gcgattaaga gtcgcgagga gggtttgcct gagggctgtg cggcttgctt cgataagcac 1620
tgctatcgtc agagctga 1638
<210> 12
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 磷脂酶变体154的多肽的氨基酸序列
<400> 12
Ala Pro Ala Pro Gly Ala Val Pro Val Pro Pro Leu Tyr Ala Glu Gly
1 5 10 15
Val Leu Glu Leu Ala Lys Arg Gln Ser Gly Leu Thr Arg Tyr Gln Pro
20 25 30
Val Glu Val Pro Cys Pro Asp Thr Ala Leu Val Arg Asp Ala Thr Gly
35 40 45
Leu Gly Thr Pro Glu Ala Glu Tyr Ile Ala Ser Arg Lys Glu Lys Ala
50 55 60
Asp Ala Ala Leu Ala Gln Trp Leu Glu Arg Gln Gly Ser Phe Gly Asp
65 70 75 80
Ser Ser Gln Pro Thr Val Ala Leu Ala Ser Ser Gly Gly Gly Tyr Arg
85 90 95
Ala Leu Leu Ala Gly Ala Gly Val Ile Lys Ala Phe Asp Ala Arg Glu
100 105 110
Gly Ser Asn Glu Val Ser Gly Leu Tyr Gln Ala Leu Thr Tyr Gln Ser
115 120 125
Gly Leu Ser Gly Gly Ala Trp Leu Leu Ser Ser Gln Ser Gly Asn Asp
130 135 140
Trp Pro Thr Ile Ser Tyr Leu Gln Glu Asn Leu Trp Glu Asp Ala Phe
145 150 155 160
Glu Val Ser Leu Leu Leu Pro Ala Asn Leu Leu Ser Phe Ser Gly Leu
165 170 175
Thr Gln Tyr Gly Ala Ile Thr Thr Glu Ile Leu Ser Lys Glu Gly Ala
180 185 190
Gly Phe Asp Thr Thr Ile Ile Asp Pro Trp Gly Arg Leu Leu Ser Tyr
195 200 205
Gln Leu Leu Glu Gly Ala Phe Gly Gly Val Asp Thr Lys Met Ser Thr
210 215 220
Leu Ser Ser Leu Ser Asn Phe Thr Asp His Asn Val Pro Phe Pro Ile
225 230 235 240
Ile Thr Ala Leu Gly Val Val Ala Arg Glu Asn Gln Cys Thr Pro Pro
245 250 255
Asp Asp Ala Thr Gln Tyr Glu Phe His Pro Tyr Glu Phe Gly Ser Trp
260 265 270
Asp Asp Gly Leu Ser Ala Phe Ala Asn Val Glu Phe Leu Gly Ser Asn
275 280 285
Leu Asn Asn Gly Gln Pro Glu Asp Ala Ser Gln Cys Val Ala Glu Tyr
290 295 300
Asp Asn Leu Gly Tyr Val Leu Gly Thr Ser Ser Asn Ile Phe Pro Ala
305 310 315 320
Ile Cys Glu Pro Leu Ser Pro Ala Asn Ser Thr Asp Asp Pro Leu Ala
325 330 335
Asn Val Leu Glu Gly Ile Ile Ser Leu Ala His Glu Pro Val Phe Glu
340 345 350
Asp Leu Phe Ala Leu Tyr Arg Asn Pro Phe Phe Asn Leu Ser Trp Ser
355 360 365
Ser Arg Val Ser Gln Asp Glu Glu Leu Leu Leu Leu Asp Gly Gly Phe
370 375 380
Ser Asn Gln Asn Val Pro Ile Trp Pro Phe Ile Val Glu Ala Arg Asp
385 390 395 400
Val Asp Val Leu Ile Val Asn Asp Asn Ser Ala Asp Thr Glu Asp Asn
405 410 415
Tyr Pro Asn Gly Thr Ala Pro His Gln Thr Tyr Leu Gln Ala Gln Leu
420 425 430
Asn Gly Leu Ala Lys Met Pro Phe Val Pro Asp Val Asn Thr Phe Val
435 440 445
Ser Gln Gly Leu Asn Gln Arg Ala Thr Phe Phe Gly Cys Asp Glu Pro
450 455 460
Asp Thr Thr Phe Ile Val Tyr Leu Pro Asn Arg Glu Tyr Ser Phe Ala
465 470 475 480
Ser Gly Gln Ser Thr Gly Lys Val Val Tyr Ser Lys Asp Glu Thr Arg
485 490 495
Ala Met Ile Ala Asn Gly Val Gln Val Gly Thr Gln Asp Gly Glu Glu
500 505 510
Gly Trp Gly Leu Cys Leu Ala Cys Ala Ile Lys Ser Arg Glu Glu Gly
515 520 525
Leu Pro Glu Gly Cys Ala Ala Cys Phe Asp Lys His Cys Tyr Arg Gln
530 535 540
Ser
545
<210> 13
<211> 545
<212> PRT
<213> 人工序列
<220>
<223> 磷脂酶变体
<220>
<221> MISC_FEATURE
<222> (182)..(182)
<223> Xaa是丙氨酸、缬氨酸、亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、
精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺
<220>
<221> MISC_FEATURE
<222> (221)..(221)
<223> Xaa是丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸
、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸和天冬酰胺
<220>
<221> MISC_FEATURE
<222> (274)..(274)
<223> Xaa是谷氨酸或丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、谷氨酸、
赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰
胺
<220>
<221> MISC_FEATURE
<222> (323)..(323)
<223> Xaa是天冬氨酸或丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨
酸、赖氨酸、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷
氨酰胺
<220>
<221> MISC_FEATURE
<222> (339)..(339)
<223> Xaa是丙氨酸、缬氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸
、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺
<220>
<221> MISC_FEATURE
<222> (342)..(342)
<223> Xaa是丙氨酸、缬氨酸、亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸、
精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺
<220>
<221> MISC_FEATURE
<222> (367)..(367)
<223> Xaa是赖氨酸、或组氨酸或丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸
、天冬氨酸、谷氨酸、赖氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬
酰胺或谷氨酰胺
<220>
<221> MISC_FEATURE
<222> (500)..(500)
<223> Xaa是缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸、天冬氨酸、谷氨酸、赖氨酸
、精氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺
<400> 13
Ala Pro Ala Pro Gly Ala Val Pro Val Pro Pro Leu Tyr Ala Glu Gly
1 5 10 15
Val Leu Glu Leu Ala Lys Arg Gln Ser Gly Leu Thr Arg Tyr Gln Pro
20 25 30
Val Glu Val Pro Cys Pro Asp Thr Ala Leu Val Arg Asp Ala Thr Gly
35 40 45
Leu Gly Thr Pro Glu Ala Glu Tyr Ile Ala Ser Arg Lys Glu Lys Ala
50 55 60
Asp Ala Ala Leu Ala Gln Trp Leu Glu Arg Gln Gly Ser Phe Gly Asp
65 70 75 80
Ser Ser Gln Pro Thr Val Ala Leu Ala Ser Ser Gly Gly Gly Tyr Arg
85 90 95
Ala Leu Leu Ala Gly Ala Gly Val Ile Lys Ala Phe Asp Ala Arg Glu
100 105 110
Gly Ser Asn Glu Val Ser Gly Leu Tyr Gln Ala Leu Thr Tyr Gln Ser
115 120 125
Gly Leu Ser Gly Gly Ala Trp Leu Leu Ser Ser Gln Ser Gly Asn Asp
130 135 140
Trp Pro Thr Ile Ser Tyr Leu Gln Glu Asn Leu Trp Glu Asp Ala Phe
145 150 155 160
Glu Val Ser Leu Leu Leu Pro Ala Asn Leu Leu Ser Phe Ser Gly Leu
165 170 175
Thr Gln Tyr Gly Ala Xaa Thr Thr Glu Ile Leu Ser Lys Glu Gly Ala
180 185 190
Gly Phe Asp Thr Thr Ile Ile Asp Pro Trp Gly Arg Leu Leu Ser Tyr
195 200 205
Gln Leu Leu Glu Gly Ala Phe Gly Gly Val Asp Thr Xaa Met Ser Thr
210 215 220
Leu Ser Ser Leu Ser Asn Phe Thr Asp His Asn Val Pro Phe Pro Ile
225 230 235 240
Ile Thr Ala Leu Gly Val Asp Ala Arg Glu Asn Gln Cys Thr Pro Pro
245 250 255
Asp Asp Ala Thr Gln Tyr Glu Phe His Pro Tyr Glu Phe Gly Ser Trp
260 265 270
Asp Xaa Gly Leu Ser Ala Phe Ala Asn Val Glu Phe Leu Gly Ser Asn
275 280 285
Leu Asn Asn Gly Gln Pro Glu Asp Ala Ser Gln Cys Val Ala Glu Tyr
290 295 300
Asp Asn Leu Gly Tyr Val Leu Gly Thr Ser Ser Asn Ile Phe Pro Ala
305 310 315 320
Ile Cys Xaa Pro Leu Ser Pro Ala Asn Ser Thr Asp Asp Pro Leu Ala
325 330 335
Asn Val Xaa Glu Gly Xaa Ile Ser Leu Ala His Glu Pro Val Phe Glu
340 345 350
Asp Leu Phe Ala Leu Tyr Arg Asn Pro Phe Phe Asn Leu Ser Xaa Ser
355 360 365
Ser Arg Val Ser Gln Asp Glu Glu Leu Leu Leu Leu Asp Gly Gly Phe
370 375 380
Ser Asn Gln Asn Val Pro Ile Trp Pro Phe Ile Val Glu Ala Arg Asp
385 390 395 400
Val Asp Val Leu Ile Val Asn Asp Asn Ser Ala Asp Thr Glu Asp Asn
405 410 415
Tyr Pro Asn Gly Thr Ala Pro His Gln Thr Tyr Leu Gln Ala Gln Leu
420 425 430
Asn Gly Leu Ala Lys Met Pro Phe Val Pro Asp Val Asn Thr Phe Val
435 440 445
Ser Gln Gly Leu Asn Gln Arg Ala Thr Phe Phe Gly Cys Asp Glu Pro
450 455 460
Asp Thr Thr Phe Ile Val Tyr Leu Pro Asn Arg Glu Tyr Ser Phe Ala
465 470 475 480
Ser Gly Gln Ser Thr Gly Lys Val Val Tyr Ser Lys Asp Glu Thr Arg
485 490 495
Ala Met Ile Xaa Asn Gly Val Gln Val Gly Thr Gln Asp Gly Glu Glu
500 505 510
Gly Trp Gly Leu Cys Leu Ala Cys Ala Ile Lys Ser Arg Glu Glu Gly
515 520 525
Leu Pro Glu Gly Cys Ala Ala Cys Phe Asp Lys His Cys Tyr Arg Gln
530 535 540
Ser
545
<210> 14
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 标签序列
<400> 14
Arg Arg Arg Arg Arg
1 5
<210> 15
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> His-Tag
<400> 15
His His His His His His
1 5
<210> 16
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 标签序列
<400> 16
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 17
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 标签序列
<400> 17
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 18
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 标签序列
<400> 18
Trp Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
Claims (10)
1.一种多肽,其特征在于,所述多肽选自:
(1)在SEQ ID NO:2所示氨基酸序列第182、221、274、323、339、342、369和500位中的至少一个位置上具有取代突变的多肽;和
(2)由(1)所示的多肽与促进(1)所示多肽表达与纯化的多肽组成的多肽。
2.如权利要求1所述的多肽,其特征在于,
所述多肽至少在第339位具有取代突变,并任选地在选自第182、274、323和500位的一个、两个或三个位置上具有取代突变;
优选地,所述多肽在第182、323和339位存在取代突变;或
优选地,所述多肽在第274、339和500位存在取代突变;或
所述多肽仅在第342位存在取代突变;或
所述多肽在第221和367位存在取代突变。
3.如权利要求1或2所述的多肽,其特征在于,所述多肽选自:
(1)氨基酸序列如SEQ ID NO:13所示的多肽;
(2)氨基酸序列如SEQ ID NO:6、8、10或12所示的多肽;
(3)由(1)或(2)所述多肽与信号肽、末端延伸、GST、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签、和/或Xa因子或凝血酶或肠激酶的蛋白水解酶位点中的一条或多条序列组成的多肽。
4.一种多核苷酸序列,其特征在于,所述多核苷酸序列选自:
(1)编码权利要求1-3中任一项所述的多肽的多核苷酸;
(2)与(1)所述的多核苷酸序列互补的多核苷酸;和
(3)(1)或(2)所述的多核苷酸的长10-40个碱基的片段;
优选地,所述多核苷酸的序列如SEQ ID NO:5、7、9或11所示。
5.一种多核苷酸构建物,其特征在于,所述多核苷酸构建物含有权利要求4所述的多核苷酸序列;
优选地,所述多核苷酸构建物是表达载体或克隆载体。
6.一种遗传工程化的宿主细胞,其特征在于,所述宿主细胞:
(1)表达权利要求1-3中任一项所述的多肽;和/或
(2)含有权利要求4所述的多核苷酸序列或权利要求5所述的多核苷酸构建物。
7.一种组合物,其特征在于,所述组合物含有权利要求1-3中任一项所述的多肽和任选的辅料,优选的,所述辅料为选自活性炭、氧化铝、硅藻土、多孔陶瓷、多孔玻璃的吸附材料。
8.权利要求1-3中任一项所述的多肽、权利要求4所述的多核苷酸序列、权利要求5所述的核酸构建物、权利要求6所述的遗传工程化的宿主细胞或权利要求7所述的组合物在油脂精炼、磷脂改性、饲料改良剂、食品工业和医药工业中的应用。
9.一种酶法脱胶方法,其特征在于,所述方法包括将权利要求1-3中任一项所述的多肽或权利要求7所述的组合物与毛油混合,进行脱胶的步骤;
优选地,所述脱胶包括在50~60℃下搅拌1~5小时,然后升温至80~90℃保持1~10分钟;
优选地,以毛油重量计,所述多肽的添加量为50~1000ppm,优选为100~500ppm,更优选为100~300ppm;
优选地,在将多肽加入毛油前,先将毛油加热至50~70℃,优选50~60℃;
优选地,所述毛油选自:大豆油、葵花籽油、花生油、菜籽油、米糠油、玉米油、橄榄油、棕榈油、棕榈仁油、棕榈软脂、卡诺拉油、蓖麻油、椰子油、芫荽油、棉籽油、榛子油、大麻籽油、亚麻籽油、芒果仁油、白芒花油、牛蹄油、红花油、山茶花油、妥尔油、椿油和其他植物油。
10.一种水解磷脂的方法,其特征在于,该方法包括使含磷脂的成分与权利要求1-3中任一项所述的多肽接触的步骤;
优选地,所述接触条件包括:
(A)温度为37~70℃,优选为50±5℃;和/或
(B)pH为3.7~7.0,优选3.4~5.8,更优选4.5±0.5;
优选地,所述磷脂是大豆磷脂。
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