Using pcr to identify bacterial strains owning high lipase activity based on their single nucleotide polymorphisms - Nguyen Quynh Uyen

TÓM TẮT Các lipase (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) là lớp enzyme xúc tác các phản ứng thủy phân lipid, phân hủy các chất dưới tác dụng của axit (acidolysis), phản ứng tạo hoặc chuyển vị ester trong các điều kiện khác nhau. Các lipase, đặc biệt là các lipase được sinh tổng hợp từ vi sinh vật, được quan tâm đặc biệt do những ứng dụng của chúng trong nhiều lĩnh vực. Tuy nhiên, cho đến thời điểm hiện tại, những phương pháp chuẩn độ (cơ chất sử dụng là dầu olive, p-nitrophenylpalimitate hay phenyl acetate) để xác định hoạt độ lipase có giá thành cao và/hoặc tốn nhiều thời gian. Trong nghiên cứu này, một phương pháp thích hợp để nhận biết cùng lúc nhiều chủng có hoạt độ lipase cao đã được thiết lập. Cụ thể, sáu đoạn trong gen mã hóa cho lipase có các mẫu hình SSCP khác nhau và ở các chủng vi sinh vật có hoạt độ lipase khác nhau đã được quan sát thấy trong số 20 chủng vi sinh vật có hoạt tính lipase. Các đoạn này được nhân dòng trong vector pGEM T Easy để xác định trình tự nucleotide của chúng và sau đó sẽ xác định các SNP có trong các đoạn này. Mồi sử dụng trong PCR được thiết kế dựa trên các SNP được xác định và sau đó mồi mới thiết kế này cùng với mồi đã được công bố được sử dụng trong phản ứng PCR cho 20 chủng vi sinh vật nêu trên. Kết quả sơ bộ cho thấy 15 chủng trong số 20 chủng nghiên cứu có sản phẩm PCR và trong 15 chủng này thì có 11 chủng (73,3%) có hoạt độ lipase lớn hơn 3,25 Unit/ml.

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TAP CHI SINH HOC 2015, 37(1se): 61-66 DOI: 10.15625/0866-7160/v37n1se. USING PCR TO IDENTIFY BACTERIAL STRAINS OWNING HIGH LIPASE ACTIVITY BASED ON THEIR SINGLE NUCLEOTIDE POLYMORPHISMS Nguyen Quynh Uyen1*, Nguyen Ha Xuyen1, Phan Thi Ha1, Nguyen Huynh Minh Quyen1, Vo Thi Thuong Lan2 1Institute of Microbiology and Biotechnology, VNU Hanoi, *uyennq@vnu.edu.vn 2Faculty of Biology, Hanoi University of Science, Vietnam National University ABSTRACT: Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are a class of enzyme that can catalyze lipolytic, acidolysis, esterification or trans-esterification under different conditions. Lipases, especially from microbial resources, have been of great interest due to their applications in many fields. However, so far the titrimetric methods, such as the methods using olive oil, p-nitrophenylpalimitate or phenyl acetate as a substrate, are too expensive and/or time consuming. In this study, a convenient method that can identify many samples with high lipase activity was established. In particular, six fragments of the strains with different patterns of single-strand conformation polymorphism SSCP were observed among 20 bacterial strains owing lipase activity. These fragments were cloned into pGEM-T Easy vector in order to sequence their nucleotides and then identify their SNPs. The PCR primer set was designed based on the SNPs of the lipase sequences and afterward this newly designed primer and published one were used to screen the 20 strains via PCR. The primary result showed that 15/20 strains gave a positive result of PCR product and 11 out of these 15 strains (73.3%) had high lipase activity (more than 3.25 Unit/ml). Keywords: Lipase activity, single strain conformation polymorphism (SSCP), single nucleotide polymorphism (SNP), PCR INTRODUCTION PCR-SSCP (Polymerase chain reaction single-strand conformation polymorphism) has proven to be a powerful approach for investigating the diversity of organisms and has proven to be useful in the identification of species and strains in the absence of distinguishing morphological characters [4]. This method could be effective to visually display different sequence types between amplicons (of 100-500 bp) and to rapidly screen a hundred samples per day [5]. PCR-SSCP has been used as a method to reliably detect Single Nucleotide Polymorphisms (SNPs) in multifactorial diseases. SNPs have been extensively studied recently as the third generation molecular marker due to their high number, stability, wide distribution and convenience for high-throughput detection method [13]. Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are a class of enzyme that can catalyze lipolytic, acidolysis, esterification or trans-esterification under different conditions [14]. Although several methods have been developed to determine lipase activity, but only two methods, i.e. the titrimetric method by using olive oil as a substrate and the colorimetric method by using p-nitrophenylpalimitate as a substrate, are commonly used in literatures. Recently, a new colorimetric method by using phenyl acetate has been developed [1]. However, all chemical methods used to determine lipase activity are expensive due to the cost of their chemicals, and they are also time consuming as the activity can be determined by only one sample at a time. In this study, 20 bacterial strains owning lipase activity were chosen. After performing PCR-SSCP of a fragment of about 260 bp, six fragments of the strains with different patterns of SSCP and with different lipase activities were cloned into pGEM-T Easy vector in order to sequence their nucleotides and then identify their SNPs. The PCR primer was designed based on the SNPs of the lipase sequences and afterward this new designed primer and published one were applied to 20 above strains via PCR. The primary result showed that 15/20 strains showed a positive result of PCR product and 11 out of these 15 strains (73.3%) had high lipase activity (more than 3.25 Unit/ml). MATERIALS AND METHODS Growth of bacteria and isolation of genomic DNAs The strains were plated on agar plates containing LB medium. Then single colonies were grown on LB liquid media and afterward their cell pellets were collected in order to extract the genomic DNAs following the protocol of Sambrook et al. (2001) [8]. Primers and PCR reaction Table 1. Primers Primers Role Source F: 5’ CCRATMRTWYTNGTNCAYGG 3’ Amplification of a fragment (around 260 bp) of lipase gene Bell Philip et. al., (2002) [2] R: 5’ AGGCCNCCCAKNGARTGNSC 3’ Lip MF: 5' AATTSYKWCAGGYGRWYCRG 3' DNA marker of lipase gene This study Lip MR: 5’ AGGCCNCCCAKNGARTGNSC 3’ Bell Philip et. al., (2002) [2] PCR reaction of 25 µl was composed of 0.5 µl DNA (~100 ng); 1 µl of forward primer (10 pmol), 1 µl of reverse primer (10 pmol); 2.5 µl MgCl2 (25 mM); 2.5 µl of 10x PCR bufer; 2 µl dNTPs (2.5 mM); 0.25 µl Taq DNA polymerase (5 U/µl); 14.25 µl H2O. PCR programe was: 95ºC: 3 s, followed by 30 cycles (95ºC: 30 s; 55ºC: 30 s and 72ºC: 1 min) and terminated by 72ºC: 10 min. After checking and purifying, the PCR products were cloned into pGEM T Easy (Promega) vector following the introduction of manufactory. Transformants were checked by PCR colony and by cutting with EcoRI to confirm the insert. The plasmids contained a fragment of 260 bp of lipase gene were sent to Macrogen (Korea) in order to determine their nucleotide sequences. SSCP 5 µl of individual PCR product were mixed with 25 µl of denaturing buffer (95% formamide, 20mM ethylenediaminetetra acetic acid (EDTA), and 0.05% bromophenol blue). The mixture was heated at 96ºC for 10 min and then chilled immediately with ice. Denatured PCR products were loaded on to 8% acrylamide-bisacrylamide non-denaturing gel with 8 ml of 40% acrylamide/bis, 4 ml of 10X TBE (89 mM Tris borate, 89 mM boric acid, 20 mM EDTA, and pH 8.0), 40 µl of tetramethylenediamine, 400 µl of 10% ammonium persulfate (APS) and 28 ml of water. Denatured PCR products were electrophoresed in prechilled 1X TBE buffer at 150 V for 6 h at 4oC. After electrophoresis, polyacrylamide gels were peeled from the glass plates and SSCP pattern was seen after staining with ethidium bromide solution. Determination of lipase activity Lipase activity is determined by using titrimetric method (5% olive oil as a substrate and 1% gum arabic, 40ºC, pH 8) [7]. One lipase activity unit is an enzyme to liberate 1 µmol of fatty acid in 1 min at certain condition. Determination of SNP The free software CLUSTAW 2.1 was used to determine the SNP of a DNA fragments. RESULTS AND DISCUSSION PCR The strains possessing lipase activity were used to extract their genomic DNAs that were subsequently used as templates to amplify the lipase fragment of about 260 bp. All parameters of PCR were optimized to get only one specific band after checking the PCR products on 1% agarose gel. The PCR representative result was shown in fig. 1. Fig. 1. PCR representative result of the fragments of the gene encoding for lipase (--): negative control of PCR, in which water was used as atemplate; M: DNA Fastruler marker; 27, 228, 11, RS1, 179 and H2: the PCR products of 8 representative strains owning lipase activity. The result showed that only one specific band with the size of around 260 bp was observed on the agarose gel. Then the PCR products were subjected to study their SSCP patterns. SSCP results and SNP After doing SSCP analysis, we found that 20 strains with different lipase activity showed6 different SSCP patterns (fig. 2). Fig. 2. Six different SSCP patterns of the 6 fragments of the lipase gene Six DNA fragments representative of these SSCP patterns were cloned into pGEM T Easy vector in order to identify their nucleotide sequences. Using the free software CLUSTAW 2.1, alignments of the lipase nucleotide sequences from 6 samples were performed, revealing differences of their nucleotides (fig. 3). The SNPs of the 6 samples were 10.5%. Figure 3. SNPs of the 6 samples with different SSCP patterns Relevance between lipase activity and the PCRs used a primer designed based on the SNPs Among the 6 samples above, strains 11 and 27 had a relatively high lipase activity and the rest of these strains had lower ones. Therefore, based on the underlined nucleotide sequence that is different in SNPs (fig. 3), the forward primer was designed and combined with the reverse published one (table 1) to re-amplify lipase fragments from 20 tested strains. We found that 15/20 strains showing a positive result of PCR product (fig. 4). The relevance between the presence of the PCR products and lipase activity of these 15 strains was determined. Notably, among these 15 strains with the positive results of PCR, 11 strains (73.3%) possessed a relatively high lipase activity of more than 3.25 Unit/ml (data not shown). Figure 4. Results of PCR reaction M: DNA marker; 46.2, M2, 747, M23, 37, 5, 29, 11, 30, 27, 156, 16, 19, 9X, 8049: the strains with positive results of PCR; (-): negative control DISCUSSION In our study, we tried to find out a convenient way to detect bacterial strains with high lipase activities via PCR by using the forward new designed primer and the published reverse one. The forward primer was designed in this study based on the SNPs of the fragments of lipase gene sequences. In this preliminary study, the evaluation of the primer was carried out on 20 strains owning lipase activity via PCR reactions. 15 out of these 20 strains showed the positive results of PCR reaction; 11/15 were with high lipase activities (more than 3.25 Unit/ml) and this corresponded to 73.3%. Notably, the PCR products were of different sizes. Probably it was due to the high diversity of the nucleotide sequences encoding lipase [10, 11, 14]. Lipases, especially from microbial resources, have been of great interest. However, the methods to determine lipase activity are too expensive and/or time consuming so far. Therefore establishing a method that can identify many samples with high lipase activity in the same time is the aim of our research. Gene polymorphisms and enzyme activity were reported to be relevant [3]. To obtain single-nucleotide polymorphisms (SNPs) by the economical and reliable way, PCR-SSCP was chosen to primarily determine the bacterial strains with different nucleotide sequences of lipase fragments. PCR-SSCP of the 16S rDNA has been used to detect plant pathogenic bacteria [9]. Markers using DNAs have played an important role in selection processes. PCR products amplified by using the primers specific for some genes (fnbB gene and SCCmec type III) were used as markers to identify strong biofilm forming of Staphyloccoccus aureus [6]. The percentage showing the relevance between PCR positive results of these genes and strong biofilm forming of the bacteria varied from 37% to 91%. In another study of Zautner, the percentage, showing the relevance between epidemiological association of Campylobacter jejuni with pathogenicity-associated genetic marker, was from around 32% to 100% [12]. In our study, this percentage is 73.3%. However, the sample numbers used in this study were not high because this was only a primary research. Therefore the validation of the primers needs to be carried out on a larger number of bacterial samples in order to confirm the relavance between high lipase activity and the nucleotide polymorphism sequence of the gene encoding lipase. CONCLUSION This study showed that based on the SNPs of lipase sequences of strains owning high and low lipase activities, the primer pair could be designed and applied to PCR to identify roughly the strains with high enzyme activity. This would provide a usefull way to initially screen many strains with high lipase activity in order to minimize the cost and the time in the selection procedure. Acknowledments: Our study is supported by the grant “Genetic evaluation of the some bacterial and actinomycetes resources of Vietnam” from MOST. REFERENCES Abd-Elhakeem A. M., Elsayed A., Alkhulaqi T., 2013. New Colorimetric Method for Lipases Activity Assay in Microbial Media. Am. J. Anal. Chem., 4: 442-444. Bell P. J. L., Sunna A., Gibbs M. D., Curach N. C., Nevalainen H., Bergquist P. L., 2002. Prospecting for novel lipase genes using PCR. Microbiol., 148: 2283-2291. Carpi M. F., Vincenzetti S., Ubaldi J., Pucciarelli S., Polzonetti V., Micozzi D., Mignini F., Napolioni V., 2013. CDA gene polymorphism and enzyme activity: genotype - phenotype relationship in an Italian - Causasian population. Future Med.: 769-781. Gasser R. B., 2006. Molecular tools - advances, opportunities and prospects. Vet. Parasitol., 136(2): 69-89. Gasser R. B., Hu M., Chilton N. B., Campbell B. E., Jex A. J., Otranto D., Cafarchia C., Beveridge I., Zhu X., 2007. Singles-strand conformation polymorphism (SSCP) for the analysis of genetic variation. Nat. Protoc., 1: 3121-3128. Lim Y., Shin H. J., Kwon A. S., Reu J. H., Park G., Kim J., 2013. Predictive genetic risk marker for strong biofilm-forming Staphylococcus aureus: fnbB gene and SCCmec type III. Diagn. Microbiol. Infect. Dis.: 539-541. Quyen D. T., Le G. T. T., Nguyen T. T., Oh T. K., Lee J. K., 2005. High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1. Prot. Exp. Purif., 39: 97-106 Sambrook J., Russell D. W., 2001. Molecular cloning: A laboratory manual, 3rd Edn, 1, New York; Cold Spring Harbor Laboratory. Srinivasa C., Sharanaiah U., Shivamallu C., 2012. Molecular detection of plant pathogenic using polymerase chain reaction single-strand conformation polymorphism. Acta Biochim. Biophys. Sin., 44: 217- 223. Vujaklija D., Schröder W., Abramíc M., Zou P., Lescić I., Franke P., Pigac J., 2002. A novel streptomycete lipase: cloning, sequencing and high-level expression of the Streptomyces rimosus GDS (L)-lipase gene. Arch Microbiol., 178: 124-130. Yuhong Z., Shi P., Liu W., Meng K., Bai Y., Wang G., Zhan Z., Yao B., 2009. Lipase diversity in glacier soil based on analysis of metagenomic DNA fragments and cell culture. J. Microbiol. Biotechnol., 19(9): 888-897. Zautner A. E., Ohk C., Tareen A. M., LugertR., Groß U., 2012. Epidemiological association of Campylobacter jejuni groups with pathogenicity-associated genetic marker. BMC Microbiol., 12: 171. Zhang H., Mao X., Zhang J., Chang X., Jing R., 2013. Single-nucleotide polymorphisms analysis of drought-resistance gene TaSnRK2.8 in common wheat. Plant Physiol. Biochem., 174-181. Zuo K., Zhang L., Yao H., Wang J., 2010. Isolation and functional expression of a novel lipase gene isolated directly from oil-contaminated soil. Acta Biochim. Pol., 57(3): 305-311. SỬ DỤNG PCR ĐỂ NHẬN BIẾT CÁC CHỦNG VI KHUẨN CÓ HOẠT ĐỘ LIPASE CAO DỰA TRÊN CÁC ĐA HÌNH ĐƠN NUCLEOTIDE Nguyễn Quỳnh Uyển1, Nguyễn Hà Xuyên1, Phan Thị Hà1, Nguyễn Huỳnh Minh Quyên1, Võ Thị Thương Lan2 1Viện Vi sinh vật và Công nghệ sinh học, ĐHQG Hà Nội 2Trường Đại học Khoa học tự nhiên, ĐHQG Hà Nội TÓM TẮT Các lipase (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) là lớp enzyme xúc tác các phản ứng thủy phân lipid, phân hủy các chất dưới tác dụng của axit (acidolysis), phản ứng tạo hoặc chuyển vị ester trong các điều kiện khác nhau. Các lipase, đặc biệt là các lipase được sinh tổng hợp từ vi sinh vật, được quan tâm đặc biệt do những ứng dụng của chúng trong nhiều lĩnh vực. Tuy nhiên, cho đến thời điểm hiện tại, những phương pháp chuẩn độ (cơ chất sử dụng là dầu olive, p-nitrophenylpalimitate hay phenyl acetate) để xác định hoạt độ lipase có giá thành cao và/hoặc tốn nhiều thời gian. Trong nghiên cứu này, một phương pháp thích hợp để nhận biết cùng lúc nhiều chủng có hoạt độ lipase cao đã được thiết lập. Cụ thể, sáu đoạn trong gen mã hóa cho lipase có các mẫu hình SSCP khác nhau và ở các chủng vi sinh vật có hoạt độ lipase khác nhau đã được quan sát thấy trong số 20 chủng vi sinh vật có hoạt tính lipase. Các đoạn này được nhân dòng trong vector pGEM T Easy để xác định trình tự nucleotide của chúng và sau đó sẽ xác định các SNP có trong các đoạn này. Mồi sử dụng trong PCR được thiết kế dựa trên các SNP được xác định và sau đó mồi mới thiết kế này cùng với mồi đã được công bố được sử dụng trong phản ứng PCR cho 20 chủng vi sinh vật nêu trên. Kết quả sơ bộ cho thấy 15 chủng trong số 20 chủng nghiên cứu có sản phẩm PCR và trong 15 chủng này thì có 11 chủng (73,3%) có hoạt độ lipase lớn hơn 3,25 Unit/ml. Từ khóa: Đa hình đơn nucleotide, hoạt độ lipase, PCR, SSCP, thủy phân lipid. Ngày nhận bài: 22-10-2014

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