WO2015127596A1 - 一种新型双功能脂肪酶突变体及其在面制品加工中的应用 - Google Patents

一种新型双功能脂肪酶突变体及其在面制品加工中的应用 Download PDF

Info

Publication number
WO2015127596A1
WO2015127596A1 PCT/CN2014/072549 CN2014072549W WO2015127596A1 WO 2015127596 A1 WO2015127596 A1 WO 2015127596A1 CN 2014072549 W CN2014072549 W CN 2014072549W WO 2015127596 A1 WO2015127596 A1 WO 2015127596A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutant
lipase
genetically engineered
amino acid
pichia pastoris
Prior art date
Application number
PCT/CN2014/072549
Other languages
English (en)
French (fr)
Inventor
徐岩
喻晓蔚
Original Assignee
江南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Priority to CN201480000483.3A priority Critical patent/CN105264070B/zh
Priority to PCT/CN2014/072549 priority patent/WO2015127596A1/zh
Priority to EP14853156.9A priority patent/EP2933330B1/en
Priority to US15/038,364 priority patent/US9890367B2/en
Publication of WO2015127596A1 publication Critical patent/WO2015127596A1/zh
Priority to US15/876,159 priority patent/US10167456B2/en
Priority to US15/876,158 priority patent/US10059931B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/109—Types of pasta, e.g. macaroni or noodles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a novel bifunctional lipase mutant and its use in the processing of pasta products, in particular to the use of molecular biology techniques to obtain lipase mutants with improved properties, which are suitable for use in the field of pasta processing. It belongs to the field of genetic engineering technology of enzymes.
  • the enzyme preparation As a protein product, based on its unique biological properties, the enzyme preparation has an irreplaceable role in baking and flour quality improvement. At present, the use of enzyme preparations in the processing of pasta products has become a mainstream trend.
  • the flour used in the bread baking process contains glycerophospholipids and triglycerides. If the glycerophospholipid is partially hydrolyzed to form lysophosphatidylphospholipids, the triglyceride is partially hydrolyzed to monoglycerides or diglycerides, lysophospholipids, monoglycerides and glycerol.
  • the diester can act as an emulsifier and has a strong gluten effect on the dough, which can improve the bread swell of the bread, increase the bread volume, make the tissue fine and uniform, soften the package, and have a better mouthfeel.
  • the flour contains lutein and lutein, which affects the whiteness of the noodle product. Therefore, if the enzyme capable of decomposing triglyceride is added to the flour to decompose the fat in the flour, the original The pigment dissolved in fat is released and is more susceptible to oxidative fading, thereby whitening the pasta.
  • the inventors successfully screened a high-yield lipase-producing strain of Rhizopus chinensis CCTCC M 201021 from the koji brewing of Luzhou-flavored Daqu liquor, and cloned the lipase gene sequence from this strain for the first time. And achieve high levels of secreted expression of the lipase in Pichia pastoris (Yu Xiao-Wei et al.
  • the amino acid sequence has the following amino acid substitutions compared to the parent amino acid sequence shown in SEQ NO. 1: Mutant 1 : P298T; Mutant 2 : P298T / H317P ; Mutant 3 : P298T / H317P/V326S ; Mutant 4 : P298T /T218S/ S234F ; mutant 5 : P298T/H317P/P168L/A129S ; mutant 6 : P298T / S234F/K161R/ V326S ;
  • the mutant has a thermal stability 14 to 38 times higher than that of the parent lipase at 40 °C.
  • Another technical problem to be solved by the present invention is to provide a genetically engineered bacteria containing the above lipase mutant and a method for constructing the same. Containing DNA encoding the lipase mutant Sequences, and genetically engineered or transgenic cell lines thereof are also within the scope of the claimed invention.
  • the genetically engineered bacteria is recombinant Pichia pastoris GS115, KM71 or SMD1168, of which GS115 is preferred. .
  • Another technical problem solved by the present invention is to provide a method for constructing the above genetically engineered bacteria by selecting a recombinant Pichia pastoris expression vector pPIC9 , pPIC3K, pPIC9K, pAO815 or pPICZ ⁇ , more preferably pPIC9 constructs a recombinant expression vector, Pichia pastoris GS115 Express for the host.
  • the amino acid substituted with 'original amino acid position' is used to indicate a mutated amino acid in a lipase mutant.
  • a mutated amino acid in a lipase mutant Such as S234F , indicating that the amino acid at position 234 is replaced by Phe of the parent lipase, and the position number corresponds to the amino acid sequence number of the parental Rhizopus oryzae lipase RCL in Table 1 of the annex sequence.
  • L180H/T128S indicates that the amino acids at position 180 and position 218 have been mutated.
  • the lipase mutant provided by the invention has the hydrolysis activity of triglyceride and lecithin, and has strong heat resistance, and has good application effect in processing of noodle products, and plays a significant whitening in the processing of dough products such as steamed bread. In the bread baking process, the effect of significantly increasing the specific volume is achieved.
  • LB liquid medium peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0.
  • MD Minimal Dextrose Medium: YNB 1.34%, Biotin 4 ⁇ 10 -5 %, Dextrose 2%, Agar 2%.
  • BMGY Breast Extract 1%, Trypton 2%, YNB 1.34%, Biotin 4 ⁇ 10 -5 %, Glycerol 1%, potassium phosphate solution pH 6.0, 100 mmol/L.
  • BMMY Breast Extract 1 %, Trypton 2 %, YNB 1.34 %, Biotin 4 ⁇ 10 -5 %, Methanol 0.5 %, potassium phosphate solution 100 mmol/L.
  • the unit in the medium is % (W/V).
  • the plasmid contains the parent Rhizopus chinensis CCTCC M201021 lipase gene ( proRCL ).
  • the plasmid containing the parental lipase gene was used as a template to obtain 5 mutant plasmids containing the following mutant amino acid sites: Mutant 1: P298T; 2: P298T / H317P; mutant 3: P298T / H317P/V326S; mutant 4: P298T /T218S/S234F; mutant 5: P298T/H317P/P168L/A129S; mutant 6 : P298T / S234F/K161R/ V326S .
  • Mutant 1 P298T
  • 2 P298T / H317P
  • mutant 3 P298T / H317P/V326S
  • mutant 4 P298T /T218S/S234F
  • mutant 5 P298T/H317P/P168L/A129S
  • mutant 6 P298T / S234F/K161R/ V326S .
  • Example 2 Construction of a genetically engineered strain expressing a lipase mutant gene
  • the mutant plasmid was transformed into colonic competent cells by heat shock method, and the positive strain was selected on the ampicillin-resistant LB plate to extract the plasmid. , sequencing verified the correct mutant plasmid.
  • the correct mutant plasmid was extracted and digested with restriction endonuclease SalI, recovered and concentrated.
  • Lipase fermentation method Recombinant Pichia pastoris is cultured in 25-50 mL BMGY medium (250 mL flask) to an OD of 2.0 to 6.0. The cells are collected by centrifugation and added to 25-50 mL of BMMY medium at 20-30. o C, continue to culture at 100-250 rpm / min, add 0.1-2.0% methanol in the volume of the culture solution every 24 h, and ferment for 72-144 h.
  • the dialysate was loaded onto SP-Sepharose FF strong positive ion exchange column chromatography (pre-equilibrated with the above buffer) ⁇ 1.6 cm ⁇ 20 cm), the unadsorbed protein was eluted with the same buffer. Afterwards, the concentration gradient of NaCl is 0-0.5 mol/L of 0.02 mol/L pH5.0. The adsorbed protein was eluted in the buffer phase of HAc-NaAc at a flow rate of 1 mL/min, and 4 mL was collected from each tube. The fractions were collected in steps to concentrate the lipase active fraction with 1.6 mol/L. Ammonium sulfate in 0.05 mol/L pH 7.5 potassium phosphate buffer was dialyzed at 4 °C for use.
  • the dialyzed enzyme solution was further chromatographed on Phenyl-Sepharose 6 FF hydrophobic column ( ⁇ 1.6 cm ⁇ 20 cm), and the equilibration buffer was buffered with 0.05 mol/L pH 7.5 potassium phosphate containing 1.6 mol/L ammonium sulfate.
  • the unadsorbed protein was removed by elution with the same buffer.
  • the adsorbed protein was eluted with a 0.05 mol/L pH 7.5 potassium phosphate buffer phase with a gradient of ammonium sulfate concentration of 0.4 mol/L, and finally eluted with H 2 O at an elution rate of 0.8 mL/min.
  • Collect 4 mL collect the fractions, concentrate the lipase active components, dialyze the salt, and freeze-dry for use.
  • the quantification of free fatty acids is as follows: Take 30 ⁇ l of the above reaction mixture and add 3ml free fatty acid quantitative reagent NEFA (Shanghai Sunshine Reagent Co., Ltd.), mix and heat at 40 ° C for 10 min.
  • Determination method of lecithin hydrolysis activity using lecithin as a substrate. Add 10ml of water to 200mg lecithin and 400 ⁇ l Triton In X-100, emulsified at 10,000 rpm for 5 min with a stirrer, 500 ⁇ l of the emulsified solution, 250 ⁇ l of 200 mM MOPS buffer (pH 6 ) and A mixture of 20 ⁇ l of 100 mM calcium chloride solution was incubated at 40 ° C for 5 min, then 40 ⁇ l of enzyme solution was added to the solution, and the mixture was incubated at 40 ° C for 10 min. Add 100 ⁇ l 1N hydrochloric acid terminates the enzyme reaction.
  • the enzyme activity is defined as pH6.0, and the reaction produces 1 ⁇ mol per minute at 40 °C.
  • the amount of fatty acid enzyme is a lipase enzyme unit.
  • Bread preparation method Refer to AACC method 10-10B and make some modifications.
  • Bread specific volume measurement determined by rapeseed exclusion method. After the bread was cooled at room temperature for 1 h, the volume and quality of the bread were measured separately.
  • Preparation method of steamed bread the formula is the raw material of steamed bread without adding any improver, 0.8% yeast (Anqi Yeast Co., Ltd.), and the amount of lipase added is 500 U/kg, no lipase was added to the blank control, and 40% water was added to mix well.
  • the above raw materials are pressed into a pressed surface (folded, pressed 6 times), hand-formed, and proofed (the formed dough is placed in a proofing box, After the process of awakening 35min, 37.5°C, humidity 80%), steam for 20min, cool and set to be tested.
  • Whiteness measurement method 8 samples to be measured are randomly determined on the hoe by a color difference meter, and the average value is taken.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Nutrition Science (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Bakery Products And Manufacturing Methods Therefor (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种新型双功能脂肪酶突变体及其在面制品加工中的应用，其氨基酸序列发生 1 ）或 2 ）形式的突变， 1 ） P298T； P298T / H317P ； P298T / H317P/V326S ； P298T /T218S/ S234F ； P298T/H317P/P168L/A129S ； P298T / S234F/K161R/ V326S ； 2 ）在 1 ）限定的核苷酸序列基础上经碱基的缺失、取代、插入或突变，同源性达到 80% 以上的核苷酸序列；该突变体在面制品加工中具有很好的应用效果，在馒头等面制品加工中起到显著增白，在面包烘焙加工中起到显著增大比容的效果，具有很好的应用前景。

Description

一种新型双功能脂肪酶突变体及其在面制品加工中的应用
技术领域
本发明涉及一种新型双功能脂肪酶突变体及其在面制品加工中的应用，具体地说涉及利用分子生物学技术获得性质提高的脂肪酶突变体，该突变体适用于面制品加工领域，属于酶的基因工程技术领域。
背景技术
酶制剂作为一种蛋白质产品，基于其独特的生物学特性，使得其在烘焙和面粉品质改良方面具有其他化学成分无法替代的作用。目前，在面制品加工中使用酶制剂已经成为一种主流的趋势。
在面包烘焙过程中使用的面粉含有甘油磷脂和甘油三脂，若将甘油磷脂部分水解生成溶血甘油磷脂，甘油三脂部分水解为甘油单酯或者甘油二酯，溶血甘油磷脂、甘油单酯和甘油二酯作为表面活性剂能够起到乳化作用，对面团有强筋作用，能够提高面包的入炉急胀，增大面包体积，使其组织细腻均匀，包心柔软，口感更好。因此在面包烘焙加工中在面粉中加入具有卵磷脂和甘油三脂分解活性的酶就能够起到上述的作用。另外在馒头等面制品加工过程中，由于面粉中含有叶黄素和叶红素等色素，影响了面制品白度，因此如果在在面粉中加入能够分解甘油三脂的酶来分解面粉中的脂肪，使得原本溶于脂肪中的色素被释放出来，更加易于被氧化褪色，从而对面制品起到增白的作用。
在前期研究中， 发明人 成功从酿造浓香型大曲酒的酒曲中筛选到一株高产脂肪酶的华根霉（ Rhizopus chinensis ） CCTCC M 201021 菌株，并从该菌株中首次克隆得到脂肪酶基因序列，并实现该脂肪酶在巴斯德毕赤酵母（ Pichia pastoris ）中的高水平分泌表达（ Yu Xiao-Wei et al. J Mol Catal B: Enzym , 2009, 57:304-311 ）；以华根霉脂肪酶基因为模板，利用定向进化技术获得了 一系列热稳定性提高的突变体（授权专利 CN 101974499B; Yu Xiao-Wei et al. Microbial Cell Factories , 2012, 11:102-112 ）。在此基础上， 本研究利用分子生物学的手段对华根霉脂肪酶进行基因突变，获得具有甘油三酯与卵磷脂水解活性的耐热脂肪酶突变体，该突变体在面制品加工中具有很好的应用效果，在馒头等面制品加工中起到显著增白、在面包烘焙加工中起到显著增大比容的效果。授权专利 CN 102160562 B 中已经公开利用本研究亲本脂肪酶作为面包添加剂提高面包的烘焙特性，本项目提供了一种性质更加优良的脂肪酶突变体，用于面包、馒头等面制品的加工工艺中。
发明内容
本发明所要解决的技术问题是提供一种新型脂肪酶突变体，其氨基酸序列如下：
1 ）所具有的氨基酸序列与 SEQ NO.1 所示亲代氨基酸序列相比，存在下列氨基酸取代形式： 突变体 1 ： P298T; 突变体 2 ： P298T / H317P ；突变体 3 ： P298T / H317P/V326S ；突变体 4 ： P298T /T218S/ S234F ；突变体 5 ： P298T/H317P/P168L/A129S ；突变体 6 ： P298T / S234F/K161R/ V326S ；
2 ）在 1 ）限定的核苷酸序列基础上经碱基的缺失、取代、插入或突变，同源性达到 80% 以上的核苷酸序列；具有甘油三酯与卵磷脂水解活性，且热稳定性好。
所述突变体的热稳定性比亲代在 40℃ 下半衰期比亲本脂肪酶高了 14~38 倍。
本发明要解决的另一个技术问题是提供一种含有上述脂肪酶突变体的基因工程菌及其构建方法。含编码所述脂肪酶突变体的 DNA 序列、及其基因工程菌或转基因细胞系亦是本发明要求保护的范围。
所述基因工程菌为重组毕赤酵母 GS115 ， KM71 或 SMD1168 ，其中优选 GS115 。
本发明解决的另一个技术问题为提供一种构建上述基因工程菌的方法，通过选取重组毕赤酵母表达载体 pPIC9 ， pPIC3K ， pPIC9K ， pAO815 或 pPICZα ，更优选 pPIC9 构建重组表达载体，以毕赤酵母 GS115 为宿主进行表达。
上述脂肪酶突变体在面制品加工中的应用也属于本发明要求保护的范围，所述面制品为面包、馒头、面条。
脂肪酶突变体的标识
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。如 S234F ，表示位置 234 的氨基酸由亲本脂肪酶的 Ser 替换成 Phe ，位置的编号对应于附件序列表 1 中亲本华根霉脂肪酶 RCL 的氨基酸序列编号。
如 L180H/ T128S ，表示位置 180 和位置 218 的氨基酸都发生了突变。
本发明提供的脂肪酶突变体具有有甘油三酯与卵磷脂水解活性，且耐热性强，在面制品加工中具有很好的应用效果，在馒头等面制品加工中起到显著增白、在面包烘焙加工中起到显著增大比容的效果。
具体实施方式
实施例中涉及到的培养基及试剂配方如下：
LB 液体培养基：蛋白胨 1 ％，酵母提取物 0.5 ％， NaCl 1 ％， pH7.0 。
MD(Minimal Dextrose Medium): YNB 1.34 ％ , Biotin 4×10-5 ％ , Dextrose 2 ％ , Agar 2 ％。
BMGY(Buffered Glycerol-complex Medium): Yeast Extract 1 ％ , Trypton 2 ％ , YNB 1.34 ％ , Biotin 4×10-5 ％ , Glycerol 1 ％ , 磷酸钾溶液 pH 6.0、 100 mmol/L 。
BMMY(Buffered Methanol-complex Medium): Yeast Extract 1 ％ , Trypton 2 ％ , YNB1.34 ％ , Biotin 4×10-5 ％ , Methanol 0.5 ％ , 磷酸钾溶液 100 mmol/L 。
培养基中的单位为％（ W/V ）。
实施例 1. 定点突变构建脂肪酶突变体表达质粒
以前期构建质粒 pPIC9K-proRCL [ 王乐乐，喻晓蔚，徐岩，华根霉 (Rhizopus chinensis ) 前导肽脂肪酶基因的克隆及其在 Pichia pastoris 中的表达， 高技术通讯， (2009) ， 19 （ 10 ） :105] 为模板，该质粒含有亲本华根霉 Rhizopus chinensis CCTCC M201021 脂肪酶基因（ proRCL ）。利用定点突变试剂盒（ QuikChange II XL Site-Directed Mutagenesis Kit, Agilent ）以含有亲本脂肪酶基因的质粒为模板，分别突变获得含有以下突变氨基酸位点的 5 个突变质粒： 突变体 1 ： P298T; 突变体 2 ： P298T / H317P ；突变体 3 ： P298T / H317P/V326S ；突变体 4 ： P298T /T218S/ S234F ；突变体 5 ： P298T/H317P/P168L/A129S ；突变体 6 ： P298T / S234F/K161R/ V326S 。
实施例 2. 表达脂肪酶突变体基因的基因工程菌的构建
将突变质粒 热激法转化大肠感受态细胞， 氨苄抗性 LB 平板上 挑选阳性菌株 ， 提取质粒 ，测序验证正确突变质粒。
提取验证正确的突变质粒，用限制性内切酶 SalⅠ 酶切，回收后浓缩。将线性化质粒与 80 μL 的毕赤酵母 GS115 感受态混匀，转至 0.2cm 电击杯中；电压 1500 V ，电容 25 µF ，电阻 200 Ω ，进行电击；电击完毕后，立即加入 1 mL 冰预冷的 1mol /L 的山梨醇溶液，混匀； 30℃ 静置 1 h ；然后涂布于 MD 平板上筛选目标基因整合到受体菌染色体上的转化子。提取酵母基因组，以 5'AOX 和 3'AOX 为引物进行 PCR 鉴定，以空载体转化菌作对照，进一步验证目的基因已经整合进酵母基因组。
实施例 3. 脂肪酶突变体的分泌表达与分离纯化
脂肪酶发酵方法：重组毕赤酵母在 25-50 mL BMGY 培养基 (250 mL 三角瓶 ) 中培养至 OD 为 2.0 ～ 6.0 ，离心收集菌体，加入 25-50 mL BMMY 培养基后于 20-30 oC ， 100-250 rpm ／ min 条件下继续培养，每 24 h 补加占培养液体积 0.1-2.0 % 的甲醇，发酵 72~144h 。
脂肪酶分离纯化方法：
(1) 10 KD 超滤膜浓缩
分别将 100 mL 发酵液 4℃ 4000 r/min 离心 20 min 后弃掉沉淀，上清液用 0.22 μm 微孔滤膜过滤，微滤后的溶液用 10 KD 超滤膜浓缩至 10 mL 左右。 浓缩酶液用 0.02 mol/L HAc-NaAc 缓冲液（ pH 5.0 ） 4℃ 透析过夜。
(2) SP-Sepharose FF 强阳离子交换柱层析
将透析液上样到已用上述缓冲液预平衡的 SP-Sepharose FF 强阳 离子交换柱层析（ Ф1.6cm×20cm ），用相同缓冲洗脱未吸附蛋白。后用 NaCl 浓度梯度为 0 ～ 0.5 mol/L 的 0.02 mol/L pH5.0 的 HAc-NaAc 缓冲液阶段洗脱吸附蛋白，流速为 1 mL/min ，每管收集 4 mL ，分步收集，将脂肪酶活性组分集中， 用 含 1.6 mol/L 硫酸铵的 0.05 mol/L pH7.5 的磷酸钾缓冲液 4℃ 透析 备用。
(3) Phenyl-Sepharose 6 FF 疏水色谱柱层析
将透析后的酶液继续用 Phenyl-Sepharose 6 FF 疏水柱层析 (Ф1.6 cm×20 cm) ，平衡缓冲液为含 1.6 mol/L 硫酸铵的 0.05 mol/L pH7.5 的磷酸钾缓冲，用相同缓冲液洗脱除去未吸附蛋白。然后用硫酸铵浓度梯度差为 0.4 mol/L 的 0.05 mol/L pH7.5 的磷酸钾缓冲液阶段洗脱吸附蛋白，最后用 H2O 洗脱，洗脱速率为 0.8 mL/min ，每管收集 4 mL ，分部收集，集中脂肪酶活性组分，透析除盐，冷冻干燥备用。
实施例 4. 脂肪酶突变体的热稳定性、甘油三脂与卵磷脂水解活性
甘油三酯水解活力的测定方法：将 10ml 水加到 200mg 橄榄油和 100mg 阿拉伯胶中，用搅拌机以 10000rpm 的条件乳化 5min ，将 200μl 乳化后的溶液、 100μl 200mM MOPS 缓冲液（ pH6 ）和 20μl 100mM 氯化钙溶液的混合液在 40℃ 保温 5min ，然后向溶液中添加 40μl 酶液，混匀后 40℃ 保温 10min 。加入 40μl 1N 盐酸终止酶反应。向该混合液中加入 400μl 4% Triton X-100 ，释放游离脂肪酸。游离脂肪酸的定量如下 : 取上述反应混合液 30μl ，加入 3ml 游离脂肪酸定量试剂 NEFA （上海阳光试剂有限公司），混匀后 40℃ 保温 10min 。
卵磷脂水解活力测定方法：以卵磷脂为底物。将 10ml 水加到 200mg 卵磷脂和 400μlTriton X-100 中，用搅拌机以 10000rpm 的条件乳化 5min ，将 500μl 乳化后的溶液、 250μl 200mM MOPS 缓冲液（ pH6 ）和 20μl 100mM 氯化钙溶液的混合液在 40℃ 保温 5min ，然后向溶液中添加 40μl 酶液，混匀后 40℃ 保温 10min 。加入 100μl 1N 盐酸终止酶反应。向该混合液中加入 400μl 4% Triton X-100 ，释放游离脂肪酸。游离脂肪酸的定量如下 : 取上述反应混合液 30μl ，加入 3ml 游离脂肪酸定量试剂 NEFA （上海阳光试剂有限公司），混匀后 40℃ 保温 10min 。
酶活的定义为 pH6.0 ， 40℃ 下反应每分钟产生 1 μmol 脂肪酸的酶量为一个脂肪酶酶活单位。
酶在 40℃ 下半衰期的测定方法：在 40℃ 下处理酶液，在不同处理时间取样，测定脂肪酶残余的甘油三酯水解活力百分比（％）。以残余酶活百分比的 log 值对时间 T （ min ）作图，直线的斜率为失活常数 k inact 。由 t 50 =ln2/k inact 得到脂肪酶在该温度下的 t 50 。
研究结果如表 1 所示，纯化的脂肪酶突变体的甘油三酯水解活力与卵磷脂水解活力比例由初始的 4.9 降低到 1.5 至 3.2 之间，表明脂肪酶突变体的卵磷脂水解活力相对脂肪酶活力有所提高。酶的热稳定性测定结果表明脂肪酶突变体在 40℃ 下半衰期比亲本脂肪酶高了 14~38 倍。
表 1 脂肪酶的酶学特性
酶 脂肪酶活力与磷脂酶活力比例（ U/mg : U/mg ） 热稳定性相对亲本脂肪酶提高倍数
亲本脂肪酶 4.9 --
突变体 1 3.2 14
突变体 2 2.7 25
突变体 3 1.8 18
突变体 4 2.9 29
突变体 5 2.2 38
突变体 6 1.5 27
实施例 5. 脂肪酶突变体在面包烘焙中的应用
在面包烘焙实验中主要考察脂肪酶制剂对面包比容的影响，并对亲本脂肪酶与突变酶的应用效果进行比较。
面包制备 方法 ：参照 AACC 法 10-10B ，并做了一定修改。面粉 100% ，食盐 1% ，白砂糖 4% ，黄油 4% ，酵母 1.5% ，水 62.5% （以小麦粉质量计），脂肪酶的添加量为 500 U/kg ，空白对照中不添加脂肪酶。将上述原料于搅拌机中搅拌 10min 后，静置 10min ，分割成 100g/ 个，搓圆，静置 10min ，成型装盘，于 38℃ 、相对湿度 85% 的条件下醒发 90min ，烘焙 25min （上火 170℃ 、下火 210℃ ），冷却后备用。
面包比容测定 ：采用菜籽排除法测定。面包在室温冷却 1h 后，分别测量面包的体积和质量。
比容（ mL/g ） = 体积（ mL ） / 质量（ g ）。
比容增加值（ % ） = （样品比容 - 空白对照比容） / 空白对照比容 *100%
研究结果表明（表 2 ） ，添加了突变脂肪酶的比容最大增加了 28% ，而亲本脂肪酶比容增加值为 21% 。显然本发明所述的脂肪酶突变体能够显著增加面包的比容，在面包烘焙中具有很好的应用价值。
表 2 脂肪酶在面制品中的应用效果
酶 相比空白对照面包比容增加值（ % ） 相比空白对照馒头白度提升单位
亲本脂肪酶 21 1.3
突变体 1 22 1.6
突变体 2 27 1.7
突变体 3 23 2.2
突变体 4 28 1.8
突变体 5 25 1.9
突变体 6 24 2.3
实施例 6. 脂肪酶突变体在馒头等面制品加工中的应用
在馒头等面制品加工中的应用实验中主要考察脂肪酶制剂对面制品白度的影响，并对亲本脂肪酶与突变酶的应用效果进行比较。
馒头制备方法：配方为未添加任何改良剂的馒头原粉， 0.8% 酵母（安琪酵母股份有限公司），脂肪酶的添加量为 500 U/kg ，空白对照中不添加脂肪酶，加水 40% 充分混合。将上述原料经过压面（面片折叠，压制 6 次）、手工成型、醒发（将成型的面团放入醒发箱， 醒发 35min 、 37.5℃ 、湿度 80% ）工序后，蒸制 20min ，冷却放置待测。
白度测定方法：用色差仪在馒头上任意的取 8 个待测定进行测定，取平均值。
研究结果表明（表 2 ），以空白对照白度提升单位记为 0 ，添加了突变脂肪酶的白度单位最大增加了 2.3 ，而亲本脂肪酶白度单位增加了 1.3 。显然本发明所述的脂肪酶突变体能够显著增加馒头面制品的白度，在馒头等面制品加工中具有很好的应用价值。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

1 、一种新型双功能脂肪酶突变体，其特征在于：
1 ）所具有的氨基酸序列与 SEQ NO.1 所示亲代氨基酸序列相比，存在下列氨基酸取代形式：
突变体 1 ： P298T;
突变体 2 ： P298T / H317P ；
突变体 3 ： P298T / H317P/V326S ；
突变体 4 ： P298T /T218S/ S234F ；
突变体 5 ： P298T/H317P/P168L/A129S ；
突变体 6 ： P298T / S234F/K161R/ V326S ；
2 ）在 1 ）限定的核苷酸序列基础上经碱基的缺失、取代、插入或突变，同源性达到 80% 以上的核苷酸序列；具有甘油三酯与卵磷脂水解活性，且热稳定性好。
2 、权利要求 1 所述脂肪酶突变体，优选 突变体 4 ： P298T /T218S/ S234F 。
3 、权利要求 1 所述脂肪酶突变体，优选 突变体 5 ： P298T/H317P/P168L/A129S 。
4 、含有权利要求 1 或 2 所述编码脂肪酶突变体基因的基因工程菌。
5 、含有权利要求 1 或 2 所述编码脂肪酶突变体基因的表达载体或克隆载体。
6 、根据权利要求 4 所述的基因工程菌，其特征在于所述基因工程菌为重组毕赤酵母 GS115 ， KM71 或 SMD1168 。
7 、根据权利要求 4 所述的基因工程菌，其特征在于所述基因工程菌为重组毕赤酵母 GS115 。
8 、权利要求 7 所述基因工程菌的构建方法，其特征在于，选取重组毕赤酵母表达载体 pPIC9 ， pPIC3K ， pPIC9K ， pAO815 或 pPICZα ，更优选 pPIC9 构建重组表达载体，以毕赤酵母 GS115 为宿主进行表达。
9 、权利要求 1 所述脂肪酶突变体在面制品加工中的应用。
10 、根据权利要求 9 所述的应用，其特征在于所述面制品为面包、馒头、面条。
PCT/CN2014/072549 2014-02-26 2014-02-26 一种新型双功能脂肪酶突变体及其在面制品加工中的应用 WO2015127596A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN201480000483.3A CN105264070B (zh) 2014-02-26 2014-02-26 一种双功能脂肪酶突变体及其在面制品加工中的应用
PCT/CN2014/072549 WO2015127596A1 (zh) 2014-02-26 2014-02-26 一种新型双功能脂肪酶突变体及其在面制品加工中的应用
EP14853156.9A EP2933330B1 (en) 2014-02-26 2014-02-26 New bifunctional lipase mutant and uses thereof in flour product processing
US15/038,364 US9890367B2 (en) 2014-02-26 2014-02-26 Bifunctional lipase mutant and methods of using same
US15/876,159 US10167456B2 (en) 2014-02-26 2018-01-21 Bifunctional lipase mutant and methods of using same
US15/876,158 US10059931B2 (en) 2014-02-26 2018-01-21 Genetically engineered Pichia strain and methods of using the same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2014/072549 WO2015127596A1 (zh) 2014-02-26 2014-02-26 一种新型双功能脂肪酶突变体及其在面制品加工中的应用

Related Child Applications (3)

Application Number Title Priority Date Filing Date
US15/038,364 A-371-Of-International US9890367B2 (en) 2014-02-26 2014-02-26 Bifunctional lipase mutant and methods of using same
US15/876,158 Division US10059931B2 (en) 2014-02-26 2018-01-21 Genetically engineered Pichia strain and methods of using the same
US15/876,159 Continuation US10167456B2 (en) 2014-02-26 2018-01-21 Bifunctional lipase mutant and methods of using same

Publications (1)

Publication Number Publication Date
WO2015127596A1 true WO2015127596A1 (zh) 2015-09-03

Family

ID=54008118

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/072549 WO2015127596A1 (zh) 2014-02-26 2014-02-26 一种新型双功能脂肪酶突变体及其在面制品加工中的应用

Country Status (4)

Country Link
US (3) US9890367B2 (zh)
EP (1) EP2933330B1 (zh)
CN (1) CN105264070B (zh)
WO (1) WO2015127596A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838704A (zh) * 2022-08-11 2023-03-24 中国海洋大学 脂肪酶突变体及其应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015127596A1 (zh) * 2014-02-26 2015-09-03 江南大学 一种新型双功能脂肪酶突变体及其在面制品加工中的应用
CN106868026A (zh) * 2017-03-22 2017-06-20 东莞泛亚太生物科技有限公司 用于生产具有耐温性脂肪酶的基因
CN110951712B (zh) * 2019-05-31 2021-07-13 江南大学 一种脂肪酶突变体及其在去污方面的应用
CN116426500B (zh) * 2023-05-18 2024-04-30 华东理工大学 一种高酯化能力的脂肪酶突变体及其表达应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974499A (zh) * 2010-08-13 2011-02-16 江南大学 热稳定性提高的脂肪酶突变体
CN102160562B (zh) 2011-03-07 2012-12-05 江南大学 利用重组华根霉脂肪酶提高烘焙特性的面包及其生产方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3037349B2 (ja) * 1989-11-17 2000-04-24 花王株式会社 酵素固定化用担体及び固定化酵素の製造方法
CN102653743B (zh) * 2010-08-13 2013-09-18 江南大学 通过定向进化构建的热稳定性提高的脂肪酶突变体
WO2015127596A1 (zh) * 2014-02-26 2015-09-03 江南大学 一种新型双功能脂肪酶突变体及其在面制品加工中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974499A (zh) * 2010-08-13 2011-02-16 江南大学 热稳定性提高的脂肪酶突变体
CN101974499B (zh) 2010-08-13 2012-07-11 江南大学 热稳定性提高的脂肪酶突变体
CN102160562B (zh) 2011-03-07 2012-12-05 江南大学 利用重组华根霉脂肪酶提高烘焙特性的面包及其生产方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
WANG LELE ET AL.: "Cloning and expression of pro- and mature Rhizopus chinensis lipase in Pichia pastoris", THE HIGH-TECH COMMUNICATIONS, vol. 19, no. 10, 2009, pages 105
WANG, RUI ET AL.: "Increasing Activity of Rhizopus chinensis CCTCC M201021 lipase by directed evolution-error prone PCR", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 25, no. 12, 25 December 2009 (2009-12-25), XP055258943 *
YU XIAO-WEI ET AL., J MOL CATAL B: ENZYM, vol. 57, 2009, pages 304 - 311
YU XIAO-WEI ET AL., MICROBIAL CELL FACTORIES, vol. 11, 2012, pages 102 - 112

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838704A (zh) * 2022-08-11 2023-03-24 中国海洋大学 脂肪酶突变体及其应用

Also Published As

Publication number Publication date
EP2933330B1 (en) 2018-12-26
US20160355794A1 (en) 2016-12-08
EP2933330A4 (en) 2016-06-15
US9890367B2 (en) 2018-02-13
US20180148700A1 (en) 2018-05-31
US10059931B2 (en) 2018-08-28
US10167456B2 (en) 2019-01-01
EP2933330A1 (en) 2015-10-21
CN105264070A (zh) 2016-01-20
US20180148699A1 (en) 2018-05-31
CN105264070B (zh) 2017-09-29

Similar Documents

Publication Publication Date Title
WO2015127596A1 (zh) 一种新型双功能脂肪酶突变体及其在面制品加工中的应用
CN110621162B (zh) 脂肪酶
JP2003515332A (ja) 生地およびパンの品質を改良する方法
CN112204136B (zh) 植酸酶突变体
CA3014326C (en) Baking lipases
EP1718159B1 (en) Preparation of dough-based product
US20230212544A1 (en) Beta-amylase enzymes
US8653241B2 (en) Phospholipase polypeptide and a DNA encoding same
JP2005508609A (ja) 酵素を使用した練り粉の調製方法
EP1989221A2 (en) Novel lipases and uses thereof
JP5918141B2 (ja) 酸性ホスホリパーゼのクローニング、発現および用途
EP3152305A1 (en) Proline-specific endoprotease and use thereof
CA3152393A1 (en) Use of gh12 cellulases in preparing bakery products comprising rye-flour
EP3152304B1 (en) Proline-specific endoprotease and use thereof
WO2023092018A1 (en) Improved enzymatic modification of galactolipids in food

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201480000483.3

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 2014853156

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14853156

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15038364

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE