Expression of glutaryl7-Aminocephalosporanic acid acylase in escherichia coli bl21(de3) and immobilization of recombinant enzyme on nanoporous materials - Vu Thi Hanh Nguyen

Journal of Science and Technology 54 (4A) (2016) 123-131 EXPRESSION OF GLUTARYL7-AMINOCEPHALOSPORANIC ACID ACYLASE IN ESCHERICHIA COLI BL21(DE3) AND IMMOBILIZATION OF RECOMBINANT ENZYME ON NANOPOROUS MATERIALS Vu Thi Hanh Nguyen, Pham Thanh Huyen, Le Gia Hy, Phi Quyet Tien * Institute of Biotechnology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam * Email: tienpq@ibt.ac.vn Received: 15 August 2016; Accepted for publication: 5 October 2016 ABSTRACT The synthesis of 7-ACA from cephalosporin C (CPC) by a two-step bioconversion using D-amino acid oxidase (DAAO) and glutaryl 7-ACA acylase (GLA) has been effectively and largely applied in pharmaceutical industry. In this study, the gene gla coding for 720-amino acid GLA from plasmid pUC57::gla was analyzed and successfully inserted into vector pET22b(+) to form expression vector pET22b(+)::gla. The newly constructed expression vector pET22b(+)::gla was cloned and then transformed into Escherichia coli BL21(DE3) to generate recombinant strain E. coli BL21(DE3)[pET22b(+)::gla]. The suitable conditions for expression of gla gene were in LB medium at 30 oC and induced by 0.4 mM of Isopropyl β-D-1- thiogalactopyranoside (IPTG) for 3 hours. Under the chosen culturing parameters, expression of gla gene by E. coli BL21(DE3)/[pET22b(+)::gla] resulted in a recombinant GLA (rGLA) with molecular weight of 83 kDa and catalytic activity of 2.7 U/mg of total protein. Experimental research on immobilization of rGLA onto ten nanoporous materials were showed that, SBA-15 was the best one for immobilization of rGLA, reaching activity of immobilized enzyme of 22.2 U/g matrix. Furthermore, optimal conditions of procedure for immobilizing rGLA on nanomaterials (SBA-15) were determined as follows: temperature is 25 C, pH7.0 and immobilization time –60 minutes. Therefore the results reported in this study revealed the successfully heterologous expression of GLA in recombinant E. coli and potential immobilization of enzyme on inorganic nano-materials. Keywords: 7-ACA, Cephalosporins, E. coliBL21(DE3)/[pET22b(+)::gla], enzyme immobilization, GLA, SBA-15. 1. INTRODUCTION 7-Aminocephalosporanic acid (7-ACA) is a very useful intermediate in the production of medically important semisynthetic cephalosporins such as cephalaglycin and cephalothin [1]. 7- ACA has been produced industrially by the deacylation of cephalosporin C (CPC) by chemical methods. Because of the environmental and safety concerns, enzymatic conversion of CPC has Vu Thi Hanh Nguyen, Pham Thanh Huyen, Le Gia Hy, Phi Quyet Tien 124 long been explored as a substitute for chemical methods [2]. The enzymatic conversion of CPC to 7-ACA was carried out by two methods [3]: (i) one-step process using cephalosporin C acylase which effectively uses CPC as substrate; however, the catalytic efficiency of these enzymes is weak thus their application is limited; (ii) two-step process comprising the conversion of CPC into GL 7-ACA, using DAAO, and its subsequent hydrolysis to 7-ACA by a GLA which have been widely applied in 7-ACA industrial production [4]. The genes coding for GLA from several Pseudomonas species have been expressed in E. coli and the enzymes were biochemically analyzed. The enzymes were found to be fully active even in a foreign host, as E. coli [5]. In E. coli, GLA was secreted into the periplasmic space [6]. In the present study, we described the cloning, nucleotide sequence and high-expression of GLA in E. coli BL21(DE3) and immobilization of recombinant enzyme on nanoporous materials. 2. MATERIALS AND METHODS 2.1. Materials The strains Escherichia coli XL1-blue and E. coli BL21(DE3) (Stratagene, USA). Plasmid pUC57::gla harboring gla gene was kindly provided by Fermentation Technology Laboratory, Institute of Biotechnology (IBT), VAST. Plasmid pET22b(+), EcoRI and SacI, ligation enzyme T4 ligase, PureLink TM - DNA purification, PureLink TM - Plasmid Extraction kits (Invitrogen, USA). Chemicals were purchased from Mecrk (Germany), Invitrogen and Fermentas (USA) and other providers. Nanoporous materials SBA-15, SBA-16, MCF and MCM were supplied by Departement of Surface Sciences and Catalysis, Institute of Chemistry, VAST. 2.2. Methods 2.2.1. Analysis of gla gene sequence in pUC57::gla: The sequence of gla gene in pUC57::gla was sequenced by using primers M13-F and M13-R in machine ABI PRISM®3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) by National Key Laboratory of Gene Technology, IBT, VAST. The sequence was analyzed by DNA-Star Software (Madison, WI, USA) and aligned by BLAST tool on NCBI (www.ncbi.nlm.nih.gov). 2.2.2. DNA transformation in E. coli by heat-shocked method: The heat-shock transformation of DNA into E. coli cells was done by method described by Sambrooket al. [7]. 2.2.3. Construction of expression plasmid pET22b(+)::gla: The plasmids pUC57::gla and pET22b(+) were dually cut by EcoRI and SacI, then gene products were purified by PureLink TM–DNA Purification (Invitrogen, USA) and ligated by T4 ligase (Invitrogen, USA). The ligation mixture of glagene and pET22b(+) were transformed into competent E. coli XL1- blue by heat-shock method and spread onto the agar-LB/amp medium. Growing transformants were then cultured in broth LB/amp and plasmid was extracted by PureLink TM–Plasmid Extraction Kit (Invitrogen, USA). The presence of glagene in newly constructed expression plasmid pET22b(+)::gla was checked by PCR using specific primers and cut by both EcoRI and SacI. The recombinant strain E. coli BL21(DE3)/[pET22b(+)::gla] was then generated by transforming vector pET22b(+)::gla into E. coli BL21(DE3). The recombinant E. coli BL21(DE3)/[pET22b(+)::gla] was used for expression of rGLA in further experiments. Expression of glutaryl 7-aminocephalosporanic acid acylase in Escherichia coli BL21 (DE3) 125 2.2.4. Expression of rGLA in E. coli BL21(DE3)/[pET22b(+)::gla]: The strain E. coli BL21(DE3)/[pET22b(+)::gla] was cultured in broth LB/amp overnight at 37 o C on rotary shaker of 220 round per munite (rpm) to prepare pre-culture. An amount of 0.5 ml of pre-culture was transferred into 5 ml of fresh LB/amp medium and cultivated at 37 o C with shaking of 220 rpm until OD600nm of culture reached up 0.6 unit. Then, the expression of rGLA was induced by isopropylthiogalactoside (IPTG) at final concentrations ranging from 0.05 - 1.0 mM. The control sample was done without induction with IPTG. To choose the harvested time, the strain E. coli BL21(DE3) was induced and grown at selected IPTG concentration and temperature. The suitable harvested time was selected by choosing the best rGLA activity expressed among samples collected at 1- 6 hours after induction. 2.2.5. Assay of rGLAactivity:Preparation of rGLA crude extract as follows: The E. coli BL21(DE3)/[pET22b(+)::gla] cells were harvested by centrifugation at 6.000 rpm for 15 min at 4 o C, washed and resuspended in 100 mM Tris HCl-100 buffer pH 8 containing 0.1 % Triton X- 100 (v/v), sonicated for 2 min and supernatant of cell lysate were used as crude extract of rGLA [7]. rGLA activity was determined by the method described by Nohair et al. [8]. 2.2.6. Immobilization of rGLA on nanomaterials: The procedure for immobilization of rGLA on nanoporous materials was performed as follows method Nohair et al.[8]. 2.2.7. Calculation of immobilization yield: Efficacy of immobilization was calculated as the ratio of the amount of protein bound on the carrier to the initial amount of protein. Yield was expressed in a percentage. The amount of protein was determined by the Bradford’s method [7]. 3. RESULTS AND DISCUSSION 3.1. Sequence of gla gene in plasmid pUC57::gla To analyze the sequence of gla gene coding for GLA, the gene inserted in plasmid pUC57::gla was sequenced. The sequencing data showed an ORF of 2,160 bp coding for 720- amino acid protein. The ORF shows high homology (about 98 – 99 %) with gla genes from Pseudomonas sp. deposited on GenBank (NCBI). Examination of recognition site by restriction enzymes found that there were no recognition sites for EcoRI and SacI. Therefore, two restriction sites EcoRI and SacI could be used for insertion of gla gene into vector pET22b(+) at MCSs positions. 3.2. Construction of expression vector pET22b(+) harboring gla gene Based on mentioned data, gene gla was cut from pUC57::gla by both enzymes EcoRI and SacI, purified and then ligated into pET22b(+) pre-treated with both EcoRI and SacI. The ligated mixture was then transformed into E. coli XL1-blue and recombinant plasmids in ten colonies of E. coli transformants were subsequently extracted. In comparison with length of native plasmid pET22b(+), all 10 extracted plasmids showed the length larger of than that of pET22b(+) without DNA insertion (Lane C). Therefore, the obtained result demonstrated that all 10 plasmids in clones could be successfully constructed by ligating gla gene into vector pET22b(+) (Fig. 1). To confirm the correct insertion of gla gene into pET22b(+), recombinant plasmids from three clones 5, 6 and 10 were randomly selected and treated with EcoRI, SacI and the presence of gla gene was checked by PCR method using Vu Thi Hanh Nguyen, Pham Thanh Huyen, Le Gia Hy, Phi Quyet Tien 126 the pair of primers GLA-F and GLA-R (Fig. 2). Random treatment of three plasmids from clones 5, 6, 10 with EcoRI or SacI (Fig. 2A) showed single DNA band of approximately 7.7 kb, corresponding to total side of gla gene (2174 bp) and plasmid pET22b(+) (5493 bp). When three plasmids (lanes 5, 6, 10) were linearized by either EcoRI or SacI, the plasmid size is about 7.7 kb which include gene gla of 2.2 kb and plasmid pET22b(+) of about 5.5 kb, respectively (Fig. 2A and 2B). The comfirmation of the presence of gla gene in three plasmids by PCR using specific primers GLA-F and GLA-R (Fig. 2B) showed single bands of 2.2 kb on lanes 7, 8, 9. Besides, the PCR amplification using pUC57::gla as DNA template also exhibited the single DNA band of 2.2 kb (lane C+) in size (Fig. 2B). Figure 1. Analysis of recombinant plasmid forming by ligation of gla gene and vector pET22b(+) on agarose gel. Lane C: Vector pET22b(+). Lane 1 to 10: Recombinant plasmids randomly extracted from E. coli XL1-blue. Figure 2. Agarose electrophoretic analysis of plasmids from three clones 5, 6, 10 cut by EcoRI and SacI (A) and Amplification of gla gene by PCR using plasmids as DNA templates (B). Lane L: Ladder 1kb; Lane 1, 2, 3: Plasmids from clones 5, 6, 10 cut by SacI; Lane 4, 5, 6: Plasmids from clones 5, 6, 10 cut by EcoRI, respectively; Lane 7, 8, 9: PCR products using plasmid from clones 5, 6, 10; Lane C - : Negative control (H2O as template); Lane C+: Positive control using pUC57::gla as the DNA template. Recombinant plasmid from the clone 5 was choosen to transfer into expression host E. coli BL21(DE3) to form recombinant E. coli BL21(DE3)[pET22b(+)::gla]. Four colonies extracted from E. coli BL21(DE3)/[pET22b(+)::gla] tranformants on agar LB/amp were then cultivated in broth LB/amp for further plasmid extraction. Four extracted plasmids were separated on agarose electrophoresis to compare the sizes with that of native plasmid pET22b(+) (data not shown). The resulting plasmids pET22b(+)::gla extracted from E. coli BL21(DE3)/[pET22b(+)::gla] were cut with each of EcoRI, SacI and used as the DNA template for amplification of gla gene by PCR (data not shown). From the obtained results, it can be concluded that recombinant vector pET22b(+)::glawas successfully transformed into expression host E. coli BL21(DE3) to form the recombinant E. coli BL21(DE3)/[pET22b(+)::gla]. The strain E. coli BL21(DE3)/[pET22b(+)::gla] was consequently used to express rGLA in next experiments. 3.3. The conditions for expression of rGLA by E. coli BL21(DE3)/[pET22b(+)::gla] 3.3.1. IPTG concentration The crude extract of rGLA from cultures of recombinant E. coli BL21 induced by different IPTG concentrations were analyzed (Fig. 3, Fig. 4). The intensity of the 83 kDa band, indicating the expression level of recombinant rGLA, from cultures induced by IPTG ranging from 0.05 - 1.0 mM showed differences in which band from 0.4 mM IPTG induction revealed the highest level (Fig. 3). In the meanwhile, the examination of rGLA activities demonstrated that rGLA 1 2 3 4 5 6 C 7 8 9 10 Expression of glutaryl 7-aminocephalosporanic acid acylase in Escherichia coli BL21 (DE3) 127 was produced at high levels in range of 0.2 to 0.6 mM IPTG induction and decreased with higher IPTG concentration of 0.6 mM. Among those, the highest rGLA activity was about 2.7 U/mg protein when the culture of E. coliBL21(DE3)/[pET22b(+)::gla] was induced by 0.4 mM IPTG. Thus, SDS-PAGE analysis of rGLA showed two bands of 54 and 16 kDa corresponding to α and β-subunit [9]. However, no distinct separation of two subunits was observed in electrophoresis results of rGLA samples induced in this study although crude extract still showed enzyme activity. Figure 3. SDS-PAGE analysis of protein crude extracts (A) and rGLA activity tests from from culture of E. coliBL21(DE3)/[pET22b(+)::gla] induced by different concentration of IPTG. Lane M: Protein marker Lane Co: Negative control using protein crude extract of E. coli BL21(DE3) Lane C1: Negative control using protein crude extract of E. coli BL21(DE3)/[pET22b(+)::gla] without IPTG induction Lane 1, 2, 3, 4, 5, 6: E. coli BL21(DE3)/[pET22b(+)::gla] induced by IPTG concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mM , respectively. Figure 4. Effect of concentration of IPTG on the rGLA activity of E. coli BL21(DE3)/[pET22b(+)::gla] 3.3.2. Incubation temperature The expression of rGLA was demonstrated to depend on the growth temperature [10]. To investigate the effect of growth temperature on rGLA productivity, the culture of recombinant E. coli BL21 was induced with 0.4 mM of IPTG at the range of temperatures of 25 o C, 30 o C, 32 o C and 37 o C for harvest times of 3 hours. As shown in Table 1, the yield of rGLA was dependent on the growth temperature with the highest activity (reaching up 109.5 %) compared with that expressed at 25 o C. In the frame of this study, the harvest times for expression of rGLA at various temperatures were selected according to previous literatures reported. The highest activity of rGLA was reached at 30 o C. 3.3.3. Harvested time for expression of rGLA To investigate the effect of harvested time on rGLA productivity, the culture of the strain E. coliBL21(DE3)/[pET22b(+)::gla] were induced by 0.4 mM of IPTG, incubated at 30 o C and shaking 200 rpm. Activities of rGLA were compared with that harvested after 4 hours of induction at 30 °C (calculated as 100 %) (Fig. 5). rGLA activity was increased strongly after 1, 2 and 3 hours of induction at 30 o C and reached the highest level of 102 % after 3 - 4 hours. Moreover, E. coli BL21(DE3)/[pET22b(+)::gla] harvested time after 5 and 6 hours were slightly decreased, revealing that prolonged incubation time did not increase the rGLA productivity. The Vu Thi Hanh Nguyen, Pham Thanh Huyen, Le Gia Hy, Phi Quyet Tien 128 suitable time for rGLA synthesis in E. coli BL21(DE3)/[pET22b(+)::gla] was 3 hours after induction. Table 1. Relative activity of rGLA in cultures expressed at different temperatures. Figure 5. Effects of harvest time on the rGLA productivity of E. coli BL21(DE3)/ [pET22b(+)::gla]. 3.4. Suitable nanomaterials for immobilization of rGLA Crude extract of rGLA was used directly for immobilization onto nanomaterials. Initial input parameters of rGLAactivity and protein concentration were fixed at start values of 2.7 U/mg protein and 4.02 mg protein/ml, respectively. Results of examination for immobilization of rGLA on ten kinds of nanoporous materials (SBA-15, SBA-15-VTES, SBA-15-PTMS, SBA- 16-M4, SBA-16-S5-APTES, MCF1, MCF2, MCF6-APTES MCM41 and MCM41-APTS) were shown in Table 2. On the basis of data in Table 2, there were significant differences in the yields of rGLA immobilized on materials among groups of SBA-15, SBA-16, MCF and MCM. The activities of immobilized GLA were high when using supporting materials SBA-15, reaching acylase activities 22.2 U/g material. Table 2. Summary of the activity of rGLA immobilized on different nanomaterials. No Nanoporous material Efficacy of immobilization*(%) Specific activity** (U/mg protein) Enzymatic activity*** (U/g material) 1 SBA-15 27.6 0.56 22.2 2 SBA-15-VTES 10.4 1.4 14.1 3 SBA-15-PTMS 5.9 0.94 7.8 4 SBA-16-M4 36.1 0.28 14.6 5 SBA-16-S5-APTES 5.56 2.1 15.6 6 MCF1 29.7 0.38 14.4 7 MCF2 8.7 1.0 12.0 8 MCF6. APTES 2.7 0.92 3.5 9 MCM41 1.8 2.2 5.2 10 MCM 41-APTS 15.2 1.0 21.4 Note: VTES - Vinyl triethoxysilane; PTMS – Phenyl trimethoxysilane; APTES – amino propyl triethoxysilane; APTS - amino propyl triethoxysilane. * % protein immobilized after immobilization; ** Enzymatic activity per 1mg of immobilized protein; *** enzymatic activity per 1 g dry support materials 3.5. Appropriate conditions for immobilization of rGLA on SBA-15 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.0 20.0 40.0 60.0 80.0 100.0 120.0 0 1 2 3 4 5 6 Ce ll w et w ei gh t ( g) Re la tiv e ac tiv ity o f G L- 7- A CA a cy la se (% ) Harvest time (hours) Activity compared with standard Cell weight Expression of glutaryl 7-aminocephalosporanic acid acylase in Escherichia coli BL21 (DE3) 129 Enzyme immobilized process is influenced by several factors such as temperature, time and pH of buffer solution [1]. The most suitable temperature for immobilization of rGLA on SBA-15 capillary is 25 o C (Fig. 6). At this temperature, enzyme activity and protein immobilized performance were the highest, reach to 23.2 U/g and 31.2 % protein. Therefore, choosing the optimal temperature for rGLA immobilization on SBA-15 is 25 ° C for next studies. At pH alkaline pH 9 - 10, the ability of immobilized rGLA on SBA-15 was not high, only 13.3 and 5.6 U/g, but optimum pH is pH 7.0 (reach to 31.2 U/g and 49.9 % protein) (Fig. 7). Meanwhile, the team of Park and Lee used silica gel or resins like amino polysiloxane, ethylene diaminepolysiloxane for GLA immobilization in phosphate buffer pH 8.0 [1, 11]. Immobilization time ofrGLA presented in Figure 8 showed that the immobilized enzyme activity was highest after 60 minutes reach to 36.6 U/g material and protein immobilized efficiency 39.5 %. If we continue to extend the time, the immobilized enzyme activity declined sharply, only 7.4 U/g after 330 minutes. The results of another study showed that the GLA immobilized on silica gel (180 minutes) or organic resin (240 minutes), it takes more time than this enzyme immobilized on SBA-15 [1, 11]. Figure 6.Effect of temperature for GLA immobilization on SBA-15. Figure 7.Effect of pH for GLA immobilization on SBA-15. 4. CONCLUSION By expression of gla gene in E. coli BL21(DE3), the recombinant GLA was successfully synthesized in LB medium at 30 o C and induced by 0.4 mM of IPTG for 3 hours. Biochemical analysis showed that the recombinant enzyme has molecular weight of about 83 kDa and specific activity of 2.7 U/mg of protein. Among of nanoporous materials for immobilization examination of rGLA, SBA-15 was the most appropriate material. The suitable conditions of procedure for immobilizing rGLA on SBA-15 were determined as follows: temperature is 25 C, pH - 7.0 and immobilization time -60 minutes (reaching the highest activity was 36.6 U/g material). Acknowledgements. This research was supported by the National Foundation for Science and Technology Development, Vietnam (NAFOSTED) under the project code 106.03-2011.07. REFERENCES 1. Park S. W., Lee J., Hong S. I. and Kim S. W. - Enhancement of stability of GL 7-ACA acylase immobilized on silica gel modified by epoxide silanization, Proc. Biochem. 39 (2003) 359-366. Figure 8.Effect of immobilization time to GLA on SBA-15. Vu Thi Hanh Nguyen, Pham Thanh Huyen, Le Gia Hy, Phi Quyet Tien 130 2. Lee Y. H., Chang T. S., Liu H. J., and Chu W. S. - An acidic glutaryl-7- aminocephalosporanic acid acylase from Pseudomonas nitroreducens, Biotechnol. Appl. Biochem. 28 (1998) 113-118. 3. Sonawane V. C. - Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes, Crit Rev Biotechnol 26 (2006) 95-120. 4. Volonte F., Marinelli F., Gastaldo L. et al. - Optimization of glutaryl-7- aminocephalosporanic acid acylase expression in E. coli, Protein Expr Purif. 61 (2008) 131-137. 5. Lee Y. S., Park S. S. - Two-step autocatalytic processing of the glutaryl 7- aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16, J. Bacteriol.180 (1998) 4576-4582. 6. Matsuda A., Komatsu K. 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TÓM TẮT NGHIÊN CỨU BIỂU HIỆN GENE GLUTARYL-7-AMINOCEPHALOSPORANIC ACID ACYLASE TRONG CHỦNG ESCHERICHIA COLI BL21(DE3) VÀ CỐ ĐỊNH ENZYME TÁI TỔ HỢP TRÊN VẬT LIỆU MAO QUẢN NANO Vũ Thị Hạnh Nguyên, Phạm Thanh Huyền, Lê Gia Hy, Phí Quyết Tiến* Viện Công nghệ sinh học, Viện Hàn lâm KHCNVN, 18 Hoàng Quốc Việt, Cầu Giấy, Hà Nội * Email: tienpq@ibt.ac.vn Quá trình sản xuất của 7-ACA từ cephalosporin C tự nhiên thông qua chuyển hóa sinh học gồm 2 bước sử dụng D-amino acid oxidase (DAAO) và glutaryl-7-ACA (GL-7-ACA) acylase Expression of glutaryl 7-aminocephalosporanic acid acylase in Escherichia coli BL21 (DE3) 131 (GLA) đã được ứng dụng hiệu quả và rộng rãi trong công nghiệp dược phẩm. Trong nghiên cứu này, gene gla mã hóa cho trình tự 720 amino-acid của GLA từ plasmid pUC57::gla đã được phân tích và chèn vào vector pET22b(+) tạo vector biểu hiện pET22b(+)::gla. Điều kiện thích hợp cho sự biểu hiện của rGLA là trong môi trường LB ở 30 °C và cảm ứng bởi 0,4 mM IPTG trong 3,0 giờ. Với điều kiện lên men đã chọn, sự biểu hiện của gene gla bởi E. coliBL21(DE3)/[pET22b(+)::gla] tạo ra GLA tái tổ hợp với khối lượng phân tử khoảng 83 kDa và hoạt tính enzyme đạt 2,7 U/mg protein tổng số. Nghiên cứu quá trình cố định rGLA trên 10 loại vật liệu mao quản nano cho thấy, SBA-15 là tốt nhất để cố định rGLA, hoạt tính enzym cố định đạt 22,2 U/g vật liệu. Hơn nữa, đã nghiên cứu điều kiện tối ưu để cố định rGLA từ E. coli tái tổi hợp trên vật liệu mao quản nano: nhiệt độ 25 °C, pH 7,0 và thời gian cố định là 60 phút. Kết quả trong nghiên cứu này cho thấy biểu sự hiện GLA trong E. coli đã thành công và tiềm năng cố định của enzyme nên các vật liệu nano. Từ khóa: 7-ACA, Cephalosporins, E. coliBL21(DE3)/[pET22b(+)::gla], enzyme cố định, GL-7- ACA acylase, SBA-15.