TG 16 high-speed centrifuge (193 10 g, Changsha Yingtai instrument co., ltd.); UV？ 2000 UV-Vis Spectrophotometer (Unocal Shanghai Instrument Factory); Vertex 70 infrared spectrometer (Brook, Germany); Me? 3250B magnetic stirring incubator (Tianjin Otsanz Instrument Co., Ltd.).
Mesoporous molecular sieve SBA? 15 was synthesized by using the surfactant pouronic p123 (EO 20po70eo20, Aldrich Company, USA) as the template and concentrated hydrochloric acid (35% ~ 37%) as the catalyst. Ethyl orthosilicate (Si(OC2H5)4, TEOS, 95.0%, Junsei Chemical Company. The synthesis methods of mesoporous molecular sieves numbered Lu00 1 and LLSD 1 are basically the same, but the raw materials are slightly different. Their BET specific surface area is 762 m2/g, pore volume is 0.87 cm3/g, pore size is 7.65438±0.8nm, and wall thickness is 3.55nm ... Candida lipase (CRL) is purchased from Amano Enzyme Technology Company in Japan. BCA protein quantitative kit was purchased from Pierce Company, USA. The experimental water is secondary distilled water.
2.2 Fourier transform infrared (FT? IR) test
The sample and KBr were dried in vacuum at 1 15℃10 h. 300 mg KBr and 2.2 mg sample were mixed and ground into fine powder in a mortar, and then pressed into tablets. After drying, they were immediately put into the in-situ cell of infrared spectrometer for testing. The resolution of the instrument is 2 cm- 1, the scanning wave number ranges from 4000 to 400 cm-1,and the scanning times are 128 times.
2.3 CRL in SBA? Fixed on 15
Centrifuge CRL phosphate buffer solution (pH 7.0) at 3000 r/min for 65438±05min, and collect supernatant to obtain collagenase solution. What about the right amount of SBA? Put 15 into collagenase solution, stir and adsorb 2 10000 r/min at 150 ~ 200 r/min in a water bath at 10000 r/min, and then centrifuge10000 r/min. Wash the molecular sieve with phosphate buffer for 4 times to elute loosely attached enzymes, centrifuge the eluent at 10000 r/min 15 min, and take the supernatant as the cleaning solution. Determine the content of enzyme protein in the original enzyme solution, raffinate solution and cleaning solution, calculate the fixed amount of enzyme protein (Mg) according to the material balance, and take the fixed amount of enzyme protein per unit mass of molecular sieve as the enzyme loading (Mg/g).
2.4 leakage of immobilized CRL
The enzyme-carrying SBA mentioned above? Transfer 15 into 70 mL phosphate buffer solution, stir in 15℃ water bath at 150 ~ 200 r/min, and take samples regularly. The samples were centrifuged at 10000 r/min 15 min, and the leakage of enzymes and protein in the supernatant was analyzed.
2.5 protein quantitative method
The content of protein in the sample was determined by single-wavelength ultraviolet spectrophotometry, dual-wavelength ultraviolet spectrophotometry and BCA method respectively. The formula of single wavelength ultraviolet method is: c (protein) (g/L) =F×A280×D/d, where A280 is the absorbance at the wavelength of 280 nm, d is the dilution multiple of the solution, d is the thickness (cm) of the cuvette, and f is the correction factor. Warburg, dual wavelength ultraviolet method? The Christian formula is: c (protein) (g/l) =1.55a280-0.76a260; Laurie? The formula of Kalckar is: c (protein) (g/l) = 1.45A280-0.74A260, where A260 and A280 are absorbance at 260 nm and 280 nm respectively. The BCA method is determined according to the protein quantitative method of Pierce Company in the United States. Analytical Chemistry Vol.37 No.8 Shang Yan et al: Mesoporous Molecular Sieve SBA? Determination of Lipase by Solid Quantitative Analysis 15 3. 1 protein Comparison of Quantitative Methods
Figure 1 shows that CRL solutions with different concentrations have a strong absorption peak at 260~280 nm, which is the characteristic peak of aromatic amino acids in protein, and is used for the determination of protein content. The CRL solution with crude enzyme concentration of 6 g/L was diluted at different times to obtain a series of enzyme solutions with known relative concentrations. The concentration of enzyme was determined by single-wavelength and dual-wavelength ultraviolet method and BCA method respectively, and whether the measured concentration ratio was consistent with its relative concentration was verified, so that the accuracy of each detection method was obtained. The results in Figure 2 show that the determination result of BCA method is the closest to the relative concentration of diluted samples, the determination value of dual-wavelength ultraviolet method is close to that of BCA method, and the determination result of single-wavelength method is much higher than that of BCA method and dual-wavelength ultraviolet method. According to the calculation results of relative concentration in table 1, the relative error of BCA method is the smallest, and the relative error of single-wavelength and dual-wavelength ultraviolet methods is larger. This is because the principle of BCA method is that Cu2+ in CuSO4 chelates protein molecules and undergoes color reaction to test absorbance, so it has strong anti-interference ability and high accuracy. Ultraviolet spectrophotometry has few operation steps, is simple and rapid, does not need chromogenic agent and does not consume samples. However, the direct detection of optical density value is greatly affected by the interference of impurities in the solution, and the error is large.
In order to investigate the effect of mesoporous molecular sieve on absorbance, 0. 1, 0.6 and 0.9 mg SBA? 15 (without Lu00 1), take distilled water as reference sample, determine its absorbance, and calculate the concentration deviation that may be caused by protein determination. Table 2 shows SBA? 15 has obvious ultraviolet absorption. Therefore, the sample solution in this experiment was centrifuged at 10000 r/min to eliminate the interference of mesoporous molecular sieve on absorbance. Table 1 Determination of protein Concentration by Different Methods Table 2 SBA? Effect of 15 on absorbance Table 3 shows SBA determined by different quantitative methods. The fixed amount of CRL is 15 (without Lu00 1) and the initial crude enzyme concentration of three parallel experiments is 6 g/L, SBA? The dosage of 15 carrier is 0.36 g, and the determination result by dual-wavelength ultraviolet method is slightly higher than that by BCA method. The results of single-wavelength ultraviolet method are much higher than those of dual-wavelength method and BCA method. Table 3 also shows that the precision of BCA method is higher than that of single-wavelength and dual-wavelength ultraviolet methods, because BCA method relies on color reaction to detect absorbance, which has high sensitivity, while mesoporous molecular sieve does not participate in color reaction, which has strong anti-interference ability and good reproducibility, and is more suitable for detecting enzyme fixation and enzyme leakage of mesoporous molecular sieve carriers; Ultraviolet spectrophotometry can be divided into three tables according to the impurities and residual mesopores in the solution: SBA? Fixed amount of CRL and enzyme loading on 15
◆: The amount of immobilized protein; ◇: amount of enzyme added. Sub-sieve has great interference. Because the results of determination of enzyme immobilization by dual-wavelength ultraviolet method are close to those by BCA method, if the experimental conditions are limited or there are few interference factors in order not to consume samples, dual-wavelength ultraviolet method can be used instead of BCA method to determine enzyme immobilization, and the interference of mesoporous molecular sieves in each sample can be offset by material balance.
3.2 BSA at different initial enzyme concentrations? Fixed amount of enzyme on 15 carrier
BCA method was used to determine the fixed amount of LLSD 1 CRL at different initial enzyme concentrations. Figure 3 shows that when the enzyme concentration is low, SBA? The fixed amount and enzyme load of 15 vector on CRL increased linearly with the increase of enzyme concentration, but when the enzyme protein concentration reached about 0.29 g/L (the crude enzyme concentration was about 1 g/L), the fixed amount and enzyme load reached a stable level, and the maximum enzyme load was114.2.
3.3 two kinds of SBA? Enzyme immobilization amount of 15 carrier
Under the same conditions that the initial enzyme concentration is 2 g/L and the molecular sieve dosage is 0. 12 g, the SEM photos of LLSD 1 and LU0 1 on CRL are 4 LLSD 1(a) and Lu00 1 (b).
The SEM images of llsd 1 (a) and lu00 1 (b) are 13.70 and 2.00mg；, respectively; The enzyme load was 1 14.2 and 16.6 mg/g, respectively. It can be seen that the fixed amount and enzyme load of LLSD 1 are much larger than that of Lu00 1. Fig. 4 shows SEM photos of LLSD 1 and Lu00 1. It can be seen that their appearance and shape are basically the same, and they all belong to SBA? There is no obvious difference in size between the traditional shapes of 15 [9]. Fig. 5 shows the FT of LLSD 1 and Lu00 1. It can be seen from the infrared spectrum that the number of hydroxyl groups on the surface of LLSD 1 is greater than that of Lu00 1. Because the adsorption of enzyme is completed by hydrogen bonding between enzyme and hydroxyl on the surface of mesoporous materials, the hydroxyl on the surface of mesoporous molecular sieve can promote the adsorption of enzyme through hydrogen bonding [10]. So, FT? Lu00 1( 1) and LLSD 1(2) in Figure 5? infra-red spectrogram
Figure 5 feet? The difference of infrared spectra between Lu 00 1 (1) and 1 (2) immobilized enzymes may be related to the hydroxyl content of mesoporous molecular sieves, and the higher the hydroxyl content, the more CRL can be immobilized.
3.4 SBA？ 15 leakage of immobilized enzyme
The immobilized enzyme is easy to "fall off" into the water phase and become a free enzyme, that is, "leakage" [1 1]. Fig. 6 shows that after 100 h, the leakage rate of the immobilized CRL of Lu00 1 in the buffer solution is 0.56%, and the leakage rate of LLSD 1 is 0.53%, indicating that the leakage amount is low. 15 is a good carrier for enzyme immobilization. The low leakage rate may be related to SBA? 15 aperture. The study [3, 12] shows that when the pore size of mesoporous materials is suitable for the size of enzyme molecules, the stability of immobilized enzyme is better. The pore size of Lu00 1 and LLSD 1 are both 7. 18 nm, and the kinetic diameter of Candida lipase is about 5 nm, which makes the enzyme molecules just fixed in the pores and not easy to leak.
◆, ■, ▲, and ● are leaks; ◇, △, ◇ are leakage rates, where ◆, ◇: 0. 12g LLSD 1, enzyme loading114.2 mg/g; ■ Mouth: 0.24 g LLSD 1, enzyme dosage11.3 mg/g; ▲, △: 0.36 g Lu00 1, enzyme load14.2 mg/g; ●, ○: 0.36 g lu00 1, enzyme loading 16.6 mg/g. 1 ray C, Shen Y, Liu J, Ackerman E J. Journal of the American Chemical Society, 2002,124:/kloc-.
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