Medium-Based Optimization of an Organic Solvent Tolerant Extracellular Lipase from The

Medium-based optimization of an organic solvent tolerant extracellular lipase from the isolated halophilic Alkalibacillus salilacusAmene Samaei-Nouroozia, Shahla Rezaeia, Nika Khoshnevisa, Mahmoud Doostib, Reza Hajihoseinia, Mohammad Reza Khoshayandc,#, and Mohammad Ali Faramarzia,* a Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran 1417614411, Iran b Department of Medical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran c Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceuticals Quality Assurance Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran------*Corresponding author: Tel: +98-21-66954712; Fax: +98-21-66954712; E-mail: [email protected] (M.A. Faramarzi)#Correspondence for statistical experimental design: Tel: +98-21-66494997; E-mail: [email protected] (M.R. Khoshayand)1 Alkalibacillus halophilus strain YIM 012 Alkalibacillus sp. A5 Alkalibacillus sp. A457 Alkalibacillus sp. M18-1 Alkalibacillus sp. LCG11 Alkalibacillus salilacus strain SR-079 Halo Alkalibacillus sp. JS20A1 Alkalibacillus salilacus strain KV001 Alkalibacillus sp. PL203 Alkalibacillus sp. HS20D Alkalibacillus sp. S1LM-8 Alkalibacillus sp. HNPhu Alkalibacillus salilacus strain R559 Alkalibacillus sp. R327 Alkalibacillus sp. BAB-4206 Alkalibacillus flavidus strain ISL-17 Bacillus sp. XJSL3-5 Alkalibacillus silvisoli strain BM2 Alkalibacillus filiformis strain 4AG Alkalibacillus haloalkaliphilus strain C5 Bacillus sp. PK-2005 Alkalibacillus sp. 15-1 Alkalibacillus sp. X1006 Tenuibacillus sp. YIM 94025 Tenuibacillus multivorans strain NBRC 100370 Filobacillus sp. MO21 Piscibacillus sp. 401C1-1 Halobacillus sp. BH178 Thalassobacillus devorans strain XJSL8-1 Microbacterium aurantiacum|Fig. S1. Tree showing the phylogenetic inter-relationships of Alkalibacillus salilacus SR-079 Halo and its closest relatives.2 1000*800 ) - 1600400 L i p a s 200 e a c t i v i t y0 ( UL p p p p p - - - - - O N N N N N l S P P P P P i v e P B D A O e s S a o u m i n l e f l o o i w l*: p-value < 0.05; [F (7,16) = 1576.81, p < 0.05]. e r o i lFig. S2. Substrate specificity of the A. salilacus SR-079 Halo lipase towards different p-NP esters and oils.3 Table S1. Minimum and maximum ranges for the parameters selected in Plackett-Burman Design.Variable Variable code -1 Value +1 ValuePeptone X1 2.75 5.00 Glucose X2 0.10 1.00 NaCl X3 1.50 3.50 MgSO4.7H2O X4 0.10 1.00 FeSO4.7H2O X5 0.10 1.00 CaCl2 X6 0.10 1.00 Olive oil X7 0.50 2.50 NH4Cl X8 1.00 10.00 KH2PO4 X9 1.00 10.00 Na2HPO4 X10 1.00 10.004 Table S2. Matrix of the Plackett-Burman design for 10 variables at two levels.Trial Variable Response 1 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 D NO. 1 +1 +1 ˗1 +1 +1 +1 ˗1 ˗1 ˗1 +1 ˗1 809.8±12.6 2 ˗1 +1 +1 ˗1 +1 +1 +1 ˗1 ˗1 ˗1 +1 709.2±7.6 3 +1 ˗1 +1 +1 ˗1 +1 +1 +1 ˗1 ˗1 ˗1 738.5±5.9 4 ˗1 +1 ˗1 +1 +1 ˗1 +1 +1 +1 ˗1 ˗1 738.5±7.3 5 ˗1 ˗1 +1 ˗1 +1 +1 ˗1 +1 +1 +1 ˗1 531.1±10.4 6 ˗1 ˗1 ˗1 +1 ˗1 +1 +1 ˗1 +1 +1 +1 732.2±12.1 7 +1 ˗1 ˗1 ˗1 +1 ˗1 +1 +1 ˗1 +1 +1 746.9±9.4 8 +1 +1 ˗1 ˗1 ˗1 +1 ˗1 +1 +1 ˗1 +1 623.3±8.9 9 +1 +1 +1 +1 ˗1 ˗1 +1 ˗1 +1 +1 ˗1 656.8±7.3 10 +1 +1 +1 +1 ˗1 ˗1 ˗1 +1 ˗1 +1 +1 719.7±5.6 11 ˗1 ˗1 +1 ˗1 +1 ˗1 ˗1 ˗1 +1 ˗1 +1 436.8±7.7 12 ˗1 ˗1 ˗1 ˗1 ˗1 ˗1 ˗1 ˗1 ˗1 ˗1 ˗1 684.0±6.4 13 0 0 0 0 0 0 0 0 0 0 0 740.6±7.2 14 0 0 0 0 0 0 0 0 0 0 0 740.6±9.1 15 0 0 0 0 0 0 0 0 0 0 0 740.6±5.1 16 0 0 0 0 0 0 0 0 0 0 0 740.6±2.3 17 0 0 0 0 0 0 0 0 0 0 0 740.6±3.05 Table S3. Design and data from the steepest ascent experiment.Trial NO. Variable ResponseGlucose NaCl Olive oil KH2PO4 1 0.55 2.50 1.50 5.50 655.8±5.4 2 0.65 2.30 1.60 5.30 717.3±8.4 3 0.75 2.10 1.70 5.10 773.3±4.0 4 0.95 1.90 1.90 4.70 852.7±3.2 5 1.15 1.70 2.10 4.30 662.7±4.1 6 1.35 1.50 2.30 3.90 648.8±1.8 7 1.55 1.30 2.50 3.50 633.5±2.56 Table S4. Experimental range and levels of the independent variables used in RSM.Variable Component Level of variable -1.68 -1 0 +1 +1.68X2 Glucose 0.16 0.50 1.00 1.50 1.84 X3 NaCl 0.82 1.50 2.50 3.50 4.18 X7 Olive oil 0.32 1.00 2.00 3.00 3.68 X9 KH2PO4 1.64 3.00 5.00 7.00 8.367 Table S5. Matrix of the Central Composite design for 4 variables at 5 levels.Trial NO. Variable ResponseGlucose NaCl Olive oil KH2PO4 1 1.50 3.50 3.00 3.00 376.4±3.5 2 1.50 3.50 1.00 3.00 788.8±6.1 3 1.50 1.50 3.00 7.00 321.9±2.0 4 0.50 3.50 1.00 7.00 649.5±6.2 5 1.50 1.50 1.00 7.00 506.6±5.0 6 0.50 1.50 3.00 3.00 794.5±3.8 7 0.50 3.50 3.00 7.00 746.1±2.9 8 0.50 1.50 1.00 3.00 319.6±0.7 9 0.20 2.50 2.00 5.00 775.4±5.3 10 1.80 2.50 2.00 5.00 762.2±4.2 11 1.00 0.82 2.00 5.00 702.7±2.6 12 1.00 4.18 2.00 5.00 856.9±4.8 13 1.00 2.50 0.30 5.00 639.6±3.7 14 1.00 2.50 3.68 5.00 509.9±3.0 15 1.00 2.50 2.00 1.64 731.6±5.0 16 1.00 2.50 2.00 8.36 636.1±0.9 17 1.00 2.50 2.00 5.00 766.8±5.2 18 1.00 2.50 2.00 5.00 767.0±4.3 19 1.00 2.50 2.00 5.00 766.8±6.1 20 1.00 2.50 2.00 5.00 767.2±7.1 21 1.00 2.50 2.00 5.00 766.8±4.28 Table S6. Analysis of variance (ANOVA results) for selected factorial model.Source Sum of Squares Df Mean Square F Value p-value Model 99007.4 4 24751.9 14.1 0.0003 X2-Glu 12524.3 1 12524.3 7.1 0.0218 X3-NaCl 24547.7 1 24547.7 14.0 0.0033 X7-Olive oil 22325.7 1 22325.7 12.7 0.0044 X9-KH2PO4 39609.8 1 39609.8 22.5 0.0006 Curvature 14182.2 1 14182.2 8.1 0.0161 Residual 19338.9 11 1758.1 14.1 Lack of Fit 19338.9 7 2762.7 0.0003 Pure Error 0 4 Cor Total 132528.5 169 Table S7. Analysis of variance (ANOVA results) for Response Surface Quadratic Model.Source Mean Squares D Mean Squares F Value p-value f Model 491357.8 14 35097.0 6.1 0.0179 X2-Glu 87.2 1 87.2 0 0.9064 X3-NaCl 11893.7 1 11893.7 2.1 0.2018 X7-Olive oil 4349.3 1 4349.3 0.8 0.4194 X9-KH2PO4 4565.5 1 4565.5 0.8 0.4087X2X3 1530.9 1 1531.0 0.3 0.6255X2X7 170666.1 1 170666.1 29.5 0.0016X2X9 3271.3 1 3271.3 0.6 0.4807X3X7 45909.1 1 45909.1 7.9 0.0305X3X9 12155.2 1 12155.2 2.1 0.1975X7X9 2829.8 1 2829.8 0.5 0.5107X2^2 8922.2 1 8922.2 1.5 0.2608X3^2 6307.7 1 6307.7 1.1 0.3368X7^2 129363.5 1 129363.5 22.3 0.0032X9^2 44350.6 1 44350.6 7.7 0.0325 Residual 34737.1 6 5789.5 Lack of Fit 34737.0 2 17368.5 494449.3 < 0.0001 Pure Error 0.1 4 0 Cor Total 526094.8 2010 Table S8. Effect of various cations on the stability of SR-079 Halo lipase. The residual activity was measured after the enzyme incubation at 40 ºC and 150 rpm for 1 h. The activity in absence of any ion was considered as 100 %.Metal ion Residual activity (%) 1 mM 5 mM 10 mM None 100 100 100 Fe3+ 147.9±1.6 152.4±1.8 98.0±0.6 Fe2+ 168.9±1.9 203.9±2.1 130.9±1.3 Ni2+ 149.4±1.5 138.9±1.7 115.0±1.2 Co2+ 91.8±0.8 84.0±0.6 68.0±0.3 Mg2+ 128.1±0.9 180.2±1.5 140.7±1.3 Mn2+ 130.5±1.2 100.9±1.1 98.4±0.7 Zn2+ 110.0±1.1 98.5±0.8 78.0±0.5 Cu2+ 148.5±1.5 178.0±1.9 134.6±1.3 Hg2+ 121.3±1.2 174.1±1.7 156.2±1.5 Al3+ 83.2±0.7 79.6±0.4 63.0±0.511 Table S9. Effect of various chemical agents on the stability of SR-079 Halo lipase. The residual activity of crude enzyme was determined after 1 h of incubation at 40 ºC and 150 rpm, and the activity without incubation in agents was defined as 100 %.Chemical agent Residual activity (%) None 100Tween 80, 0.1 % 472.3±3.1 Tween 80, 0.25% 457.9±3.8 Tween 80, 0.5% 399.0±3.5 Triton 0.1 % 317.7±3.3 Triton 0.25 % 389.9±3.4Triton 0.5 % 459.1±3.92-ME 0.1 % 316.5±3.1 2-ME 0.25 % 325.6±3.3 2-ME 0.5 % 343.0±3.4 PMSF 1 mM 118.3±0.9 PMSF 5 mM 75.3±0.7 CTAB 1 mM 193.5±1.1 CTAB 10 mM 367.1±2.3 SDS 0.1 % 59.8±0.4 SDS 0.2 % 75.3±0.7 SDS 0.5 % 87.9±0.6 EDTA 2 mM 90.9±0.8 EDTA 5 mM 93.3±0.7 EDTA 10 mM 79.3±0.6 Urea 1 mM 77.7±0.4 Urea 5 mM 128.4±1.212

Medium-Based Optimization of an Organic Solvent Tolerant Extracellular Lipase from The

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