Libraries were screened for lipase activity in microtitre plates using the synthetic chromogenic substrate,/7-nitrophenyl palmitate (PNP-palmitate).
70jul aliquots from each master plate well were replica-plated to assay wells containing ISpl of 0.5M Tris-Cl (pH 8.0), Lids were sealed to the assay plates with Parafilm and the plates incubated at 80®C for 15 minutes. 15pi of 1.5mM PNP-palmitate in ethanol were added to the assay wells and incubated at 60°C. Plates were inspected periodically for up to 8 hours. Lipase activity was indicated by the development of a yellow colour due to the production ofp-nitrophenolate.
E. coli TOPlO/pCR- and lipase (Sigma) were used as negative and positive controls, respectively.
Chapter 2 Materials & Methods
2.9.1.3: Microtitre-plate screening for phosphatase activity
Libraries were screened for phosphatase activity in microtitre plates using the synthetic chromogenic substrate, p-nitrophenyl phosphate (PNP-phosphate).
75-jul aliquots from each master plate well were replica-plated to assay wells containing 15jul of 0.5M Tris-Cl (pH 8.0). Lids were sealed to the assay plates with Parafilm and the plates incubated at 80°C for 15 minutes. To start the reaction, lOjul of 30mM PNP- phosphate in distilled H2O was added to the assay wells and the plates incubated at 60°C. Plates were inspected periodically for up to 8 hours. Phosphatase activity was indicated by the development of a yellow colour which corresponded to the production of p-nitrophenolate.
E. coli TOPlO/pCR- and phosphatase (Sigma) were used as negative and positive controls, respectively.
2.9.2: Screening libraries for enzyme activities using indicator agar plates
Environmental DNA libraries were screened for a-amylase, lipase, phosphatase and protease activities using indicator agar plates prepared as described below. Libraries were plated out at a titre of 2000 cfu per 140mm plate and incubated overnight at 37®C. Prior to enzyme detection, indicator plates were placed into sealed plastic bags, transferred to 50°C and incubated overnight.
Because incubating at 50°C kills E, coli, plasmids were prepared from these clones using a modified mini-preparation protocol for plasmid isolation (Section 2.8.14). Recovered plasmids were re-introduced into E. coli by electroporation (Section 2.8.11). The controls used in the microtitre plate assays were also used in the indicator plate assays. Protease (Sigma) was used as a positive control for the skim milk indicator plates. The enzymes were used by spotting into the agar o f the appropriate indicator plate.
2.9.2.1: Starch indicator plates for a-amylase activity (Gerhardt et al., 1981)
Indicator plates were prepared by supplementing nutrient agar with soluble starch at a final concentration of 0.25% (w/v). After the 50°C-incubation step, the starch plates
were flooded with iodine solution which was prepared by adding 1ml 2,2% I2 /4.4% KI solution to 500ml 2% (w/v) KI solution. The iodine solution was decanted as each plate became saturated with the purple-black starch/iodine complex. A halo around the colony indicated amylase activity.
2.9.2.2: Tween-80 indicator plates for lipase activity (Gerhardt et aL, 1981)
Indicator plates were prepared by supplementing nutrient agar with CaCl2 (0.01% (w/v) final concentration). After autoclaving and cooling to 50°C, Tween-80 was added to the medium at a final concentration of 1% (v/v). After the 50°C incubation step, plates were inspected for lipase activity as indicated by the occurrence of opaque haloes around lipase-positive colonies.
2.9.2.3: TPMG (tryptose phosphate-methyl green) indicator plates for phosphatase
activity (Riccio 1997)
Indicator plates were prepared by supplementing tryptose phosphate agar with phenolphthalein diphosphate (Img/ml final concentration), and methyl green (SOpg/ml final concentration). Phosphatase activity is indicated by the formation of green halos or the green staining of phosphatase-positive colonies.
2.9.2.4: Skim milk indicator plates for protease activity
Indicator plates were prepared by supplementing nutrient agar with 1% (w/v) powdered skim milk. Protease activity is indicated by the occurrence of clear zones around protease-positive colonies.
2.10: DNA Sequencing and Analysis
DNA sequences were determined by Oswel DNA services (Northampton). Nucleotide and deduced amino acid sequences obtained from environmental genomic libraries were compared with each other or with entries in the nonredundant nucleic acid and protein databases of the National Center for Biotechnology Information (NCBI) server using BLASTN or BLASTX (Altschul et a l, 1990; Gish & States, 1993).
Open reading frames were located within DNA sequences using the ORF finder at NCBI. Multiple protein sequence alignments were achieved using CLUSTAL W server
Chapter 2 Materials <è Methods
amino acid sequences were identified using FingerPRINTscan software located on the server at the University of Manchester Bioinformatics Unit (Scordis et a l, 1999). Putative bacterial promoter sequences were screened by either visually scanning regions upstream o f ORFs for consensus -35 (TTGACA) and -10 (TATAAT) hexamers (Gross
et a l, 1992) or by using online software for bacterial promoter prediction at www.fhiitfly.org/seq_tools/promoter.html (Reese et a l, 1996). Upstream sequences were also visually scanned for archaeal consensus promoter elements (Soppa, 1999). tRNA gene sequences were scanned using the tRNAscan-SE search sever at Washington University, S t Louis (Lowe & Eddy, 1997).
2.11: Thermostable Enzyme Activities in E, coli Cell Extracts
Enzymic activities of selected clones were assayed using cell extracts prepared from recombinant E. coli. Cultures were grown to stationary phase and diluted in nutrient broth to give an OD^oonm reading o f -1.5. Cells (50ml) were harvested (10 min at 4°C and SOOOx g) and resuspended in 5ml nutrient broth. The cells (1ml) were disrupted on ice using a MSE Soniprep 150 ultrasonic disintegrator (SANYO Gallenkamp Pic., Leicester). Sonication was performed at an amplitude of 8-^m for 2 min at 10-sec intervals. The extract was cleared by centrifugation at top speed in a microfuge for 10 min at 4°C. Aliquots of cell extracts were incubated at different temperatures for 15 minutes before being assayed for enzymic activities using microtitre plates or 1.5-ml microfuge tubes essentially as described in Section 2.9.2.