Preparation of lipase with improved ester synthesis activity by using surfactants

Abstract

The present disclosure discloses a method for preparing lipase with high ester synthesis activity by using surfactant, belonging to the field of enzyme engineering. The present disclosure provides a method for obtaining a lipase with high ester synthesis activity by adding different surfactants with different concentrations in a lipase aqueous solution and then freeze-drying. The lipase meets the requirement in non-aqueous catalysis. Mixing a variety of lipase with no ester synthesis activity or low activity at suitable concentration and an appropriate concentration of the surfactant in the solution can produces lipases with significantly improved ester synthesis activity, meanwhile changing the hydrolytic activity of the lipase. Increased ester synthesis activity makes lipase more suitable for industrial applications.

Claims

1. A method for increasing ester synthesis activity of lipase, comprising: adding surfactant directly to an aqueous solution of soluble lipase, and mixing, wherein the soluble lipase comprises Rhizopus chinesis lipase r27RCL, wherein the surfactant comprises n-dodecyl-β-D-maltopyranoside, and wherein the surfactant is present at a final concentration of from 1 mM to 500 mM and from 0.1 times to 200 times critical micelle concentration.

2. The method for increasing the ester synthesis activity of lipase according to claim 1, comprising dissolving the lipase in powder form directly into the aqueous solution.

3. The method for increasing the ester synthesis activity of lipase according to claim 1, wherein the aqueous solution comprises phosphate buffered saline or Tris-HCl buffer.

4. The method for increasing the ester synthesis activity of lipase according to claim 1, wherein the soluble lipase is heterologously expressed by a recombinant microorganism.

5. The method for increasing the ester synthesis activity of lipase according to claim 1, wherein protein concentration of lipase in the aqueous solution of the soluble lipase is between 0.1 mg/mL and 0.4 mg/mL.

6. A lipase preparation obtained by: dissolving the lipase in an aqueous solution; adding surfactant directly to the aqueous solution; wherein the soluble lipase comprises Rhizopus chinesis lipase r27RCL, wherein the surfactant comprises n-dodecyl-β-D-maltopyranoside, and wherein the surfactant is present at a final concentration of from 1 mM to 500 mM and from 0.1 times to 200 times critical micelle concentration; and removing water from the aqueous solution.

Description

DETAILED DESCRIPTION

(1) Determination of Lipase Activity

(2) (1) Determination of hydrolytic activity of the lipase using p-nitrophenyl palmitate (pNPP) as substrate

(3) Hydrolysis of pNPP catalyzed by lipase generates p-nitrophenol and palmitic acid. p-nitrophenol appears yellow in the buffer at pH 8.0 with the maximum absorption peak at 410 nm. Determination of hydrolytic activity can be achieved by measuring the absorbance at 410 nm.

(4) The substrate solution A: 50 mmol.Math.L.sup.−1 sodium phosphate buffer (pH 8.0) containing 1.16 g.Math.L.sup.−1 sodium deoxycholate and 0.56 g.Math.L.sup.−1 arabic gum. Substrate solution B: 0.015 g pNPP is dissolved in 5 mL isopropyl alcohol. Substrate solution A and substrate solution B are mixed then stored for use afterwards.

(5) Termination solution: 40 g.Math.L.sup.−1 NaOH, 93.05 g.Math.L.sup.−1 EDTA sodium. Add 62 uL termination solution to the reaction mixture when stopping the reaction.

(6) Determination method:

(7) Add 0.1 mL appropriately diluted enzyme solution, which is substituted by inactivated enzyme solution as the control, to 2.4 mL substrate solution above, incubate the mixture at 40° C. for 2 min and measure the absorbance at 410 nm.

(8) Definition of enzyme activity:

(9) one hydrolytic activity unit is defined as the amount of enzyme that catalyzes the formation of 1 μmol of p-nitrophenol in 1 minute at 40° C.

(10) Calculation equation:

(11) enzyme activity (U.Math.mL.sup.−1)=(V×A.sub.410×10.sup.6)/(ε×t×V′)

(12) where V is the volume of the reaction mixture (mL), ε is the molar extinction coefficient (mL.Math.mmol.sup.−1), t is the reaction time (min) and V′ is the volume of the enzyme solution (mL).

(13) (2) Determination of ester synthesis activity of the lipase by GC analysis

(14) Reaction substrates:

(15) Substrate solution A: 48.5 mL of n-octanoic acid is dissolved in 250 mL of n-heptane in a volumetric flask.

(16) Substrate solution B: 17.5 mL of absolute ethanol is dissolved in 250 mL of n-heptane in a volumetric flask.

(17) Internal standard: 2-hexanol/n-heptane (35 g.Math.L.sup.−1).

(18) Standard sample: 10 g of n-octanoate is dissolved in 1000 mL of n-heptane in a volumetric flask.

(19) Method of Determination

(20) 1 mL substrate solution A and substrate solution B are added in a 5 mL Eppendorf tube respectively, then 20 mg lipase powder (or lyophilized powder) is added. The reaction is carried out at 40° C. and with shaking at 150 rpm for 30 min. Remove the lipase powder by centrifugation or membrane filtration. Then 0.1 mL of internal standard is added in 0.4 mL of filtrate or supernatant and mixed. Measure the content of n-octanoate in the mixture above by GC analysis.

(21) The gas chromatograph (6820, Agilent Instruments) is equipped with a AC20 (PEG20000)capillary column and a FID detector. Nitrogen was used as the carrier gas. The oven temperature was programmed to start at 90° C. for 1 min and then be elevated to 200° C. for 5 min at 10° C..Math.min.sup.−1. The injector and detector temperatures were set at 250° C.

(22) Definition of enzyme activity: One unit of ester synthesis activity is defined as the amount of enzyme that esterifies 1 micromole of n-octanoate per min.

(23) Calculation equation:

(24) $\mathrm{enzyme}\mathrm{activity}\left(U.Math.m{g}^{-1}\right)=\frac{A_{\mathrm{sam}}}{A_{\mathrm{sta}}}\times S_{\mathrm{sta}}\times V\times {10}^6\times \frac{1}{172}\times \frac{1}{30}\times \frac{1}{m}$

(25) A.sub.sam—the ratio of the peak areas of the sample for testing to the internal standard;

(26) A.sub.sta—the ratio of the peak areas of the standard sample to the internal standard;

(27) S.sub.sta—the concentration of the standard sample (g˜L.sup.−1);

(28) V—the volume of the reaction mixture (L);

(29) m—the amount of lipase in the reaction mixture (mg).

(30) Specific activity: the activity of lipase per milligram of Natalprotein in the lipase preparation.

(31) Total recovery: the percentage of the activity after a treatment to that before the treatment

EXAMPLE 1

Method of Operation for Enhancing Lipase Ester Synthesis Activity

(32) Add an equal volume of high-concentration surfactant solution into the enzyme solution. The final protein concentration was 0.25 mg.Math.mL.sup.−1 and the surfactant concentration was 10 mM. After mixed thoroughly, the mixture was lyophilized and kept in dry condition.

EXAMPLE 2

Regain of the Ester Synthesis Activity of Commercial r27RCL

(33) Commercial lipase r27RCL was purchased from Jiangsu Yiming Biological Technology Co., Ltd, which has high hydrolytic activity and low ester synthesis activity. Dissolve the r27RCL powder in water and centrifuge to obtain the supernatant containing the r27RCL lipase to adjust the ester synthesis activity (the final protein concentration was 0.2 mg.Math.mL.sup.−1 and the final concentration of surfactant was 10 mM, which is between 0.1×CMC.sup.˜200×CMC of the different surfactants in Table 1). As shown in Table 1, addition of LPC14 increased the ester synthesis specific activity from 5.5 U.Math.mg.sup.−1 to 47.6 U.Math.mg.sup.−1 and the total recovery of ester synthesis activity was 769% while the hydrolytic specific activity did not show significant change.

(34) TABLE-US-00001 TABLE 1 Ester synthesis activity and hydrolytic activity of r27RCL. Specific activity Total recovery surfactant (U .Math. mg.sup.−1) (%) Ester blank 5.5 100 synthesis CTAB 0 0 activity DiC6PC 15.9 256 C12E8 22.2 372 LPC14 47.6 769 DDM 31.4 423 CHAPS 13.0 105 Triton X-100 12.9 192 Hydrolytic blank 115.7 100 activity CTAB 0 0 DiC6PC 104.2 87 C12E8 117.1 101 LPC14 116.1 97 DDM 135.0 94 CHAPS 132.6 109 Triton X-100 116.6 93

EXAMPLE 3

Regain of the Ester Synthesis Activity of Commercial ROL

(35) Commercial lipase ROL was purchased from Sigma-Aldrich Co., Ltd. ROL is a non-immobilized lipase. Dissolve the ROL powder in water and centrifuge to obtain the supernatant containing the ROL lipase that would be regulated to improve its ester synthesis activity by adjusting the final protein concentration to 0.2 mg.Math.mL.sup.−1 and adding surfactants to the final concentration of 10 mM. As shown in Table 2, addition of LPC14 increased its ester synthesis specific activity from 0.4 U.Math.mg.sup.−1 to 12.2 U.Math.mg.sup.−1 and the total recovery of ester synthesis activity was 1733%.

(36) TABLE-US-00002 TABLE 2 Ester synthesis activity and hydrolytic activity of ROL. Specific activity Total recovery surfactant (U .Math. mg.sup.−1) (%) Eester blank 0.4 100 synthesis CTAB 0 0 activity DiC6PC 3.11 733 C12E8 2.6 400 LPC14 12.2 1733 DDM 2.8 333 CHAPS 2.6 400 Triton X-100 1.7 200 Hydrolytic blank 18.5 100 activity CTAB 0 0 DiC6PC 15.2 76 C12E8 23.7 82 LPC14 33.1 106 DDM 42.7 116 CHAPS 23.6 81 Triton X-100 34.9 99

EXAMPLE 4

Regain of the Ester Synthesis Activity of Commercial PCL

(37) Commercial lipase PCL was purchased from Sigma-Aldrich Co., Ltd. PCL is a non-immobilized lipase. Dissolve the PCL powder in water and centrifuge to obtain the supernatant containing the PCL lipase that would be regulated to improve the ester synthesis activity by adjusting the final protein concentration to 0.2 mg.Math.mL.sup.−1 and adding surfactants to the final concentration of 10 mM. As shown in Table 3, addition of DDM increased its ester synthesis specific activity from 2.5 U.Math.mg.sup.−1 to 55.6 U.Math.mg.sup.−1 and the total recovery of ester synthesis activity was 1435%.

(38) TABLE-US-00003 TABLE 3 Ester synthesis activity and hydrolytic activity of PCL. Specific activity Total recovery surfactant (U .Math. mg.sup.−1) (%) Ester Blank 2.5 100 synthesis CTAB 0 0 activity DiC6PC 7.1 260 C12E8 4.6 130 LPC14 44.6 1260 DDM 55.6 1435 CHAPS 4.7 130 Triton X-100 6.3 174 Hydrolytic blank 214.8 100 activity CTAB 75.5 29 DiC6PC 308.2 140 C12E8 281.1 98 LPC14 321.8 119 DDM 327.7 111 CHAPS 248.0 101 Triton X-100 364.5 131

EXAMPLE 5

Increase of the Ester Synthesis Activity of Commercial CALB

(39) Commercial lipase CALB was purchased from Sigma-Aldrich Co., Ltd. CALB is a non-immobilized lipase. Dissolve the CALB powder in water and centrifuge to obtain the supernatant containing the CALB lipase that would be regulated to improve the ester synthesis activity by adjusting the final protein concentration to 0.12 mg.Math.L.sup.−1 and adding surfactants to the final concentration of 10 mM. As shown in Table 4, addition of DDM increased the ester synthesis specific activity from 7.8 U.Math.mg.sup.−1 to 101.9 U.Math.mg.sup.−1 and the total recovery of ester synthesis activity was 700%.

(40) TABLE-US-00004 TABLE 4 Ester synthesis activity and hydrolytic activity of CALB. Specific activity Total recovery surfactant (U .Math. mg.sup.−1) (%) Ester Blank 7.8 100 synthesis CTAB 36.6 300 activity DiC6PC 31.9 400 C12E8 62.8 467 LPC14 36.8 411 DDM 101.9 700 CHAPS 5.9 44 Triton X-100 77.4 544 Hydrolytic blank 1.1 100 activity CTAB 0 0 DiC6PC 0.98 85 C12E8 0 0 LPC14 0.54 38 DDM 0 0 CHAPS 1.6 92 Triton X-100 0 0

EXAMPLE 6

Effect of Concentration of Surfactant LPC14 on Ester Synthesis Activity of Commercial r27RCL

(41) Commercial lipase r27RCL was purchased from Jiangsu Yiming Biological Technology Co., Ltd. r27RCL is a lipase with high hydrolytic activity but low ester synthesis activity. Dissolve the r27RCL powder in water, centrifuge to obtain the supernatant and treat the supernatant with different concentrations of surfactant LPC14 (the final protein concentration was 0.2 mg.Math.mL.sup.−1). As shown in Table 5, ester synthesis activity of r27RCL obtained more improvement at higher concentration of surfactant LPC14 (100×CMC).

(42) TABLE-US-00005 TABLE 5 Effect of concentration of LPC14 on ester synthesis activity and hydrolytic activity of r27RCL. concentration Specific activity Total recovery (x CMC) (U .Math. mg.sup.−1) (%) Ester 0 5.5 100 synthesis 0.1 1.8 32 activity 1 1.4 25 100 17.1 292 Hydrolytic 0 115.7 100 activity 0.1 120.2 101 1 122.2 102 10 128.0 107 100 125.7 102

EXAMPLE 7

Effect of Concentration of Surfactant LPC14 on Ester Synthesis Activity of Commercial ROL

(43) Commercial lipase ROL was purchased from Sigma-Aldrich Co., Ltd. and is a non-immobilized lipase. Dissolve the ROL powder in water, centrifuge to obtain the supernatant and treat the supernatant with different concentrations of surfactant LPC14 (the final protein concentration was 0.2 mg.Math.mL.sup.−1). As shown in Table 6, ester synthesis activity obtained more improvement at higher concentration of surfactant LPC14 (10×100×CMC).

(44) TABLE-US-00006 TABLE 6 Effect of concentration of LPC14 on ester synthesis activity and hydrolytic activity of ROL. concentration Specific activity Total recovery (x CMC) (U .Math. mg.sup.−1) (%) Ester 0 0.4 100 synthesis 0.1 0.4 97 activity 1 0.2 46 10 1.0 240 100 6.2 1517 Hydrolytic 0 18.5 100 activity 0.1 28.7 150 1 27.7 139 10 31.2 162 100 39.0 206

EXAMPLE 8

Effect of Concentration of Surfactant DDM on Ester Synthesis Activity of Commercial PCL

(45) Commercial lipase PCL was purchased from Sigma-Aldrich Co., Ltd. and is a non-immobilized lipase. Dissolve the PCL powder in water, centrifuge to obtain the supernatant and treat the supernatant with different concentrations of surfactant DDM (the final protein concentration was 0.2 mg.Math.mL.sup.−1). As shown in Table 7, ester synthesis activity obtained more improvement at higher concentration of surfactant DDM (10×-100×CMC).

(46) TABLE-US-00007 TABLE 7 Effect of concentration of DDM on ester synthesis activity and hydrolytic activity of PCL concentration Specific activity Total recovery (x CMC) (U .Math. mg.sup.−1) (%) Ester 0 2.5 100 synthesis 0.1 3.2 58 activity 1 2.9 57 10 20.0 260 100 101.6 2776 Hydrolytic 0 214.8 100 activity 0.1 499.5 106 1 434.7 99 10 756.7 114 100 773.5 246

EXAMPLE 9

Effect of Concentration of Surfactant DDM on Ester Synthesis Activity of Commercial CALB

(47) Commercial lipase CALB was purchased from Sigma-Aldrich Co., Ltd. and is a non-immobilized lipase. Dissolve the CALB powder in water, centrifuge to obtain the supernatant and treat the supernatant with different concentrations of surfactant DDM (the final protein concentration was 0.12 mg.Math.mL.sup.−1). As shown in Table 8, ester synthesis activity obtained more improvement at higher concentration of surfactant DDM (100×CMC).

(48) TABLE-US-00008 TABLE 8 Effect of concentration of DDM on ester synthesis activity and hydrolytic activityof CALB. concentration Specific activity Total recovery (x CMC) (U .Math. mg.sup.−1) (%) Ester 0 7.8 100 synthesis 0.1 4.0 12 activity 1 14.3 50 10 37.3 73 100 32.6 189 Hydrolytic 0 1.1 100 activity 0.1 2.1 45 1 3.0 75 10 0.7 10 100 0 0

(49) In summary, the present disclosure obtains the lipase preparation with high ester synthesis activity by in vitro regulating the activity of the lipase by simulating the hydrophobic microenvironment around the protein using surfactants. The ester synthesis activity of the lipase r27RCL expressed by Pichia pastoris was increased from 5.5 U.Math.mg.sup.−1 to 47.6 U.Math.mg.sup.−1 and the total ester synthesis activity recovery was 769% after the surfactant treatment. The ester synthesis activity of commercial ROL was increased from 0.4 U.Math.mg.sup.−1 to 12.2 U.Math.mg.sup.−, and the total activity recovery was 1733% after the surfactant treatment. The ester synthesis activity of commercial PCL was increased from 2.5 U.Math.mg.sup.− to 55.6 U.Math.mg.sup.−1, and the total activity recovery was 1435% after the surfactant treatment. The ester synthesis activity of commercial CALB was increased from 7.8 U.Math.mg.sup.−1 to 101.9 U.Math.mg.sup.−1, and the total activity recovery was 700% after the surfactant treatment. The concentrations of surfactant used significantly affected the regain of lipase ester synthesis activity. The higher concentration (10×100×CMC) of the surfactant can result in better lipase ester synthesis activity. The increase of both the specific activity and total activity recovery indicated that the activity regulation by simulating the hydrophobic environment of cell membrane in vitro had a positive effect on the ester synthesis activity of lipase. This method was not only applicable to r27RCL, but also to other lipases.

(50) The disclosure described and claimed herein is not to be limited in scope by the specific aspects herein disclosed. Any person skilled in the art can make modifications without departing from the spirit and scope of the disclosure. The scope of protection of the present disclosure should therefore be defined by the claims.