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 salilacus

Amene 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

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*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 ) - 1

400 L i p a

s 200 e a c t i v i t y

L 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

Fig. 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 Value

Peptone 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 10.00

4 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±3.0

5 Table S3. Design and data from the steepest ascent experiment.

Trial NO. Variable Response

Glucose 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±2.5

6 Table S4. Experimental range and levels of the independent variables used in RSM.

Variable Component Level of variable -1.68 -1 0 +1 +1.68

X2 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 8.36

7 Table S5. Matrix of the Central Composite design for 4 variables at 5 levels.

Trial NO. Variable Response

Glucose 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±4.2

8 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 16

9 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 0.4087

X2X3 1530.9 1 1531.0 0.3 0.6255

X2X7 170666.1 1 170666.1 29.5 0.0016

X2X9 3271.3 1 3271.3 0.6 0.4807

X3X7 45909.1 1 45909.1 7.9 0.0305

X3X9 12155.2 1 12155.2 2.1 0.1975

X7X9 2829.8 1 2829.8 0.5 0.5107

X2^2 8922.2 1 8922.2 1.5 0.2608

X3^2 6307.7 1 6307.7 1.1 0.3368

X7^2 129363.5 1 129363.5 22.3 0.0032

X9^2 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 20

10 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±0.5

11 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 100

Tween 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±3.4

Triton 0.5 % 459.1±3.9

2-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±1.2