# Patent

## Title
Bifunctional lipase mutant and methods of using same

## Abstract

The present invention provides a novel dual-function lipase variant and its application in processing of flour products. The amino acid sequence of the lipase has one of the following amino acid substitutions: P298T, P298T/H317P, P298T/H317P/V326S, P298T/T218S/S234F, P298T/H317P/P168L/A129S, P298T/S234F/K161R/V326S or the nucleotide sequence of wherein said lipase is substituted, deleted, or added based on the sequence encoding the amino acid described in (a) and has at least 80% identity with it. The variants have good performance in processing of flour products, while they can significantly whiten the bread or other products in processing of flour products and significantly increase the specific volume in bread baking process.

## Background
FIELD OF THE INVENTION
The present invention relates to a novel dual-function lipase variant and its application in processing of flour product. Specially, the lipase variant with enhanced characteristics may be obtained by molecular biology techniques, which is suitable for processing of flour products. The present invention relates to the field of enzyme engineering.
BACKGROUND
Enzyme plays an irreplaceable role in improvement of baking and flour quality because of its unique biological characteristics. Currently, the usage of enzymes in processing of flour products has become a mainstream trend.
The flour used for bread baking contains glycerol phospholipids and triglycerides. When the glycerol phospholipids were partially hydrolyzed into hemolysis glycerophospholipids and triglycerides were partially hydrolyzed into monoglycerides or diglycerides, the hemolysis glycerophospholipids, monoglycerides and/or diglycerides would work as surfactants during emulsion reaction. In addition, emulsification plays a part in strengthening the dough gluten, enhancing urgent expansion in the furnace and expanding volume, which makes the bread uniform, soft and have a better taste. Therefore, addition of enzyme which has hydrolysis activity of glycerol phospholipids and triglycerides to the flour during bread baking process would obtain effects as described above. Moreover, pigments such as lutein and carotene in flour would affect the whiteness of flour products. If an enzyme which has hydrolysis activity of triglycerides is added to the flour to degrade fat, the fat-soluble pigments would be released and then fade more easily through oxidization. As a result, the flour products would be whitened.
In previous studies, the inventors successfully screened Rhizopus chinensis CCTCC M 201021 from Luzhou-flavor liquor brewing koji, which has a high yield of lipase and cloned the lipase gene from the genome for the first time and then the recombinant strain achieved high level secretion for the lipase in Pichia pastoris (Yu Xiao-Wei et al J Mol Catal B: Enzym, 2009, 57: 304-311). The inventors also used the lipase gene as the template and obtained a series of variants with improved thermostability by directed evolution (Granted patent: CN 101974499B; Yu Xiao-Wei et al. Microbial Cell Factories, 2012, 11:102-112). Then, the lipase gene from R. chinensis was mutated by molecular biology in this invention and heat resistant variants with hydrolytic activity of triglyceride and lecithin were obtained. The variants performed better at whitening bread or other products during processing of flour products and increasing the specific volume in bread baking process. As a granted patent CN 102160562 B has disclosed, the parent lipase as an additive has improved the baking characteristics of bread baking, while the present invention provides more excellent lipase variants which could be used in processing of flour products.


DETAILED DESCRIPTION
The goal of the present invention is to provide a novel lipase variant, which has one of the characteristics described as follows:
(1) whose amino acid sequence has comprises one of the substitutions relative to the parent amino acid sequence shown in SEQ ID NO.1:
mutant 1: P298T (Proline→Threonine);
mutant 2: P298T/H317P (Proline→Threonine and Histidine→Proline);
mutant 3: P298T/H317P/V326S (Proline→Threonine, Histidine→Proline and Valine→Serine);
mutant 4: P298T/T218S/S234F (Proline→Threonine, Threonine→Serine and Serine→Phenylalanine);
mutant 5: P298T/H317P/P168L/A129S (Proline→Threonine, Histidine→Proline, Proline→Leu and Alanine→Serine);
mutant 6: P298T/S234F/K161R/V326S (Proline→Threonine, Serine→Phe, Lysine→Argnine and Valine→Serine);
(2) by deletion, substitution, insertion or mutation of one or more bases, a nucleotide sequence of the lipase has at least 80% identity with a nucleotide sequence encoding the amino acid sequence described in (1), and the lipase maintains a triglyceride and lecithin hydrolysis activity and good performance in thermostability.
The lipase variant exhibits better thermostability, and its half-life is prolonged by 14-38 fold at 40° C. than the parent lipase.
The present invention also provides a genetic engineering strain harboring a sequence encoding the lipase variant and its construction. DNA sequence encoding the lipase variants and genetically engineered bacteria or transgenic cell lines harboring the DNA sequence is also in the true scope of the present invention.
The genetic engineering strain is P. pastoris GS115, KM71 or SMD1168, and preferred GS115.
The present invention also provides a method of constructing the genetic engineering strain. Expression vector pPIC9, pPIC3K, pPIC9K, pAO815 or pPICZα is selected to construct a recombinant expression vector and P. pastorisGS115 is selected as the host to express the lipase variant. More preferably, the expression vector is pPIC9.
Application of the above lipase variants in processing of flour products also belongs to the true scope of the present invention. The flour products comprise bread, steamed bread and noodles.
Nomenclature for amino acid modifications in the present invention is explained in detail as follows.
The mutated amino acid in the variant is marked as “original amino acid, position, substituted amino acids”. For example, S234F indicates a substitution of Ser in position 234 with Phe. The position number corresponds to the amino acid sequence of the parent lipase shown in SEQ ID NO: 1, and L180H/T128S indicates the position of 180 and 218 are both mutated.
The lipase variants provided in the present invention are thermostable and have triglyceride hydrolysis activity and lecithin hydrolysis activity. While they can significantly whiten the bread or other products and significantly increase the specific volume in bread baking process, the lipase variants have good application in processing of flour products.
EMBODIMENTS
Media:
LB liquid medium: 10 g·L−1 trypton, 5 g·L−1 yeast extract, 10 g·L−1 NaCl, pH7.0.
MD (Minimal dextrose medium): 13.4 g L−1 YNB, 4×10−4 g L1 biotin, 20 g·L−1 dextrose, 20 g·−1 agar.
BMGY (Buffered glycerol-complex medium): 10 g·L−1 yeast extract, 20 g·L−1 trypton, 13.4 g L−1 YNB, 4×10−4 g L−1 biotin, 10 g·L−1 glycerol, pH 6.0 100 mM potassium phosphate.
BMMY (Buffered methanol-complex medium): 10 g·L−1 yeast extract, 20 g·L−1 trypton, 13.4 g L−1 YNB, 4×10−4 g L−1 biotin, 5 g·L−1 methanol, 100 mM potassium phosphate.
Example 1 Construction of Recombinant Expression Vector by Site-Directed Mutagenesis
The pre-constructed plasmid pPIC9K-proRCL (Wang Lele et al. Cloning and expression of pro- and mature Rhizopus chinensis lipase in Pichia pastoris. The high-tech communications, (2009), 19 (10): 105) contains the lipase gene of the parent strain R. chinensis CCTCC M201021 and it was used as a template. Site directed mutagenesis kit (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent) was used to obtain the following six mutated plasmids:
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.
Example 2 Construction of Recombinant Strain Expressing the Lipase Variant
The mutated plasmid was transformed into competent cells by heat shock. The positive strains were then selected from the LB media containing ampicillin. And then the plasmids were extracted from the strains and sequenced for verification.
The extracted correct mutated plasmid was digested with restriction endonuclease Sal I and then collected and condensed. Then, the linearised plasmid was mixed with 80 μL competent P. pastoris GS115 and removed to 0.2 cm cuvette for electric shock. The shock was conducted at voltage of 1500 V with the capacitance of 25 μF and the resistance of 200Ω.
After the shock, 1 mL pre-icecold sorbitol solution with a concentration of 1 mol/L was immediately added and mixed. Then the mixture was standing at 30° C. for 1 h before applying on MD plate to screening the corrected recombinant which integrated the target gene to its chromosome. The yeast genome was extracted and amplified by primers 5′AOX and 3′AOX for PCR identification. As a negative control, the strain contained the empty vector was used to validate that the target gene had been integrated into the yeast genome.
Example 3 Expression and Secretion of the Lipase Variant and its Separation and Purification
The positive recombinant yeast was cultivated in 25-50 mL of BMGY medium in 250 mL glass flasks. When cultures reached an OD of 2.0-6.0, the cells were centrifuged and resuspended with 25-50 mL of BMMY medium shaken at 20-30° C. and 100-250 rpm for 72-144h. Methanol was added with an amount of 1% of the broth volume every 24 h.
Protein separation and purification was achieved by the following steps.
(1) Concentrated by 10 KD Ultrafiltration
100 mL broth was centrifuged at 4° C. and 4000 rpm for 20 min. Then the supernatant was filtered by 0.22 μm microporous membrane and concentrated to about 10 mL through an 10 KD ultrafiltration. The concentrated enzyme solution was interchanged overnight with 0.02 mol/L HAc-NaAc buffer (pH 5.0) at 4° C.
(2) Purified by Strong Cation Exchange Chromatography
The interchanged solution was loaded onto a pre-equilibrated SP-Sepharose FF column (1.6 cm×20 cm) and eluted with the same buffer (0.02 mol/L pH 5.0 HAc-NaAc buffer) to remove the unadsorbed protein. Then the absorded protein was eluted with 0-0.5 M NaCl in the same buffer at a flow rate of 1 mL/min. The lipase activity component was concentrated stepwise and interchanged overnight with 0.05 M potassium phosphate buffer (pH7.5) containing 1.6 M ammonium sulfate at 4° C. for use.
(3) Purified by Hydrophobic Interaction Chromatography
The interchanged enzyme solution of step 2 was then treated with a Phenyl-sepharose 6 FF column (1.6 cm×20 cm). 0.05 M potassium phosphate buffer (pH7.5) containing 1.6 M ammonium sulfate was used as the equilibration buffer and also used to elute the unadsorbed protein. Lipase was then eluted by an ammonium sulfate concentration gradient of 0.4 M in 0.05 M potassium phosphate buffer (pH7.5) and then eluted with water at a flow rate of 0.8 mL/min. And 4 mL fractions containing the activity component were collected, interchanged and then freeze-dried for use.
Example 4 Lipase Properties
(1) Hydrolysis Activity
(a) Determination of Triglyceride Hydrolysis Activity
10 mL water was added to 200 mg olive oil and 100 mg arabic gum. The mixture was then emulsified by stirring at 10000 rpm for 5 min. Next, The 200 μL emulsified solution, 100 μL 0.2 M MOPS buffer (pH 6) and 20 μl 0.1 M calcium chloride solution was mixed and held at 40° C. for 5 min. Then 40 μL enzyme solution was added, mixed and held at 40° C. for 5 min before 40 μL hydrochloric acid (1N) was added to terminate the enzymatic reaction. Finally, 400 μL 4% Triton X 100TRITON-X-100 was added to the mixture to release the free fatty acids.
(b) Determination of Lecithin Hydrolysis Activity
In this case, lecithin was used as a substrate. At first, 10 mL water was added to 200 mg lecithin and 400 μL TRITON-X-100. The mixture was then emulsified by stirring at 10000 rpm for 5 min. Next, the 500 μL emulsified solution, 250 μL 0.2 M MOPS buffer (pH 6) and 20 μL 0.1 M calcium chloride solution was mixed and held at 40° C. for 5 min. And then 40 μL enzyme solution was added, mixed and held at 40° C. for 10 min before 100 μL hydrochloric acid (1N) was added to terminate the enzymatic reaction. Finally, 400 μL 4% TRITON-X-100 was added to the mixture to release the free fatty acids.
The method of quantifying the free fatty acids was as follows. 3 mL free fatty acid quantitative reagent NEFA was added to 30 μL the above reaction mixture and then kept at 40° C. for 10 min.
One unit of enzyme activity was defined as the amount needed to release 1 μmol fatty acid per minute under 40° C. and pH 6.0.
The results were shown in Table 1. The ratio of the triglyceride hydrolysis activity and lecithin hydrolysis activity of the purified lipase variants were decreased from 4.9 to 1.5-3.2, indicating that the lecithin hydrolysis activity of the variants was increased compared to the triglyceride hydrolysis activity.
(2) Thermal Stability
The determination of half-life of the enzyme at 40° C. was carried out by the following steps. The enzyme solution was treated in 40° C. at various time intervals and the percentage (%) of residual triglyceride hydrolysis activity was determined. The time (min) was set as the X-axis and the logarithm of the percentage (%) of residual activity was set as the Y-axis. The inactivation constant kinact kinact was determined by the slope and the half-life of the enzyme (t50) at 40° C. was determined by t50=ln 2/kinact.
Results of thermal stability (Table 1) showed that the half-life of the variants at 40° C. were prolonged by 14-38 fold compared with the parent lipase.







TABLE 1







Enzymatic properties of lipase










Ratio of triglyceride
Prolonged fold of



hydrolysis activity and leci-
thermal stability



thin hydrolysis activity
compared to the


Enzyme
(U/mg:U/mg)
parent lipase





the parent lipase
4.9
—


mutant 1
3.2
14


mutant 2
2.7
25


mutant 3
1.8
18


mutant 4
2.9
29


mutant 5
2.2
38


mutant 6
1.5
27









Example 5 Application of Lipase Variants in Bread Baking
The bread baking experiments mainly focused on the impact of the lipase on the bread specific volume, and the application effect was compared between the parent lipase and the variants.
The preparation of bread was referenced to the AACC 10-10B method with a few modification. 100% flour, 1% salt, 4% sugar, 4% butter, 1.5% yeast and 62.5% water (according to the wheat mass) were mixed and lipase was added to it with an amount of 500 U/kg, while the amount of lipase in the blank control was zero. The mixture were stirred in a stirrer for 10 min, and then stood for 10 min before divided into 100 g. Next, the mixture was rounded, and stood for 10 min again. The dough was formed and put on plates and then proofed at 38° C. with a relative humidity of 85% for 90 min. Finally, the dough was baked for 25 min (upper-side temperature was 170° C. and lower-side temperature was 210° C.).
The determination of bread specific volume was carried out according to the rapeseed exclusion method. The volume and weight of the bread were measured after cooling at room temperature for 1h.
The specific volume (mL/g)=volume (mL)/weight (g).
The increased specific volume (%)=(the specific volume of sample−the specific volume of blank control)/the specific volume of blank control*100%
The results were shown in Table 2. It indicated that the maximum increased specific volume of the bread adding the lipase variant was 28%, while the parent lipase was 21%. It is clearly that the lipase variants of the present invention can significantly increase the specific volume of bread and making it a highly promising candidate for future applications in bread baking.







TABLE 2







Results of application of lipase in flour products












The increased
The increased white-




specific volume
ness compared with



Enzyme
(%)
the blank control







the parent lipase
21
1.3



mutant 1
22
1.6



mutant 2
27
1.7



mutant 3
23
2.2



mutant 4
28
1.8



mutant 5
25
1.9



mutant 6
24
2.3










Example 6 Application of Lipase Variants in Steamed Bread
The experiments mainly focused on the impact of the lipase to the whiteness of the flour products such as steamed bread. And the application effect was compared between the parent lipase and the variants.
The preparation of steamed bread was carried out according to the following method. The materials were original steamed bread powder without any improver and 0.8% yeast (Angel Yeast Co., Ltd.). The added amount of lipase was 500 U/kg, while the blank control was zero. 40% water was added to the materials above and mixed thoroughly. The mixture was pressed (the dough was pressed into pieces and folded and then pressed for six times), hand-formed, proofed (proofed in the dough proofing machine at 37.5° C. with a relative humidity of 80%) and then steamed for 20 min.
The whiteness was determined by averaging the whiteness of eight random position of the steamed bread.
The increased whiteness of the blank control was set as 0. Results (shown in Table 2) indicated that the maximum increased whiteness of steamed bread adding lipase variants was 2.3 units, while it was 1.3 in the steamed bread adding parent lipase. Obviously, the lipase variants in the present invention can significantly increase the whiteness of flour products such as steamed bread, which indicating a highly promising applications in processing of flour products.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

## Claims

1. A lipase mutant, comprising:
an amino acid sequence with substitutions selected from the group consisting of P298T, P298T/H317P, P298T/H317P/V326S, P298T/T218S/S234F, P298T/H317P/P168UA129S, and P298T/S234F/K161R/V326S, wherein the substitutions are relative to a parent amino acid sequence set forth in SEQ ID NO: 1; and the lipase mutant maintains a triglyceride and lecithin hydrolysis activity and good performance in thermostability.

2. The lipase mutant of claim 1, wherein the amino acid sequence comprises the substitution P298T/T218S/S234F, and wherein the substitutions are relative to the parent amino acid sequence set forth in SEQ ID NO: 1.

3. The lipase mutant of claim 1, wherein the amino acid sequence comprises the substitution P298T/H317P/P168L/A129S, and wherein the substitutions are relative to the parent amino acid sequence set forth in SEQ ID NO: 1.