541

Vol.56, n.4: pp. 541-546, July-August 2013 BRAZILIAN ARCHIVES OF ISSN 1516-8913 Printed in Brazil BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL

Mutation Breeding of Lipase-producing Strain Flavobacterium sp. by Supercritical CO 2 with Hydrate

Qiaoyan Zhang 1,2 , Junqing Qian 1* and Lingzhi Ma 1 1College of Pharmaceutical Science; Zhejiang University of Technology; Hangzhou, Zhejiang 310014; China. 2Institute of Quality and Standard for Agro-products; Zhejiang Academy of Agricultural Sciences; Hangzhou, Zhejiang 310021 - China

ABSTRACT

This work aimed to obtain an ideal mutant strain with higher lipase yield using hydrazine hydrate (HZH) as a novel additive to treat Flavobacterium sp. strain YY25 by supercritical CO 2. The survival rate and the positive mutation rate of the tested strain were strongly dependent on the dose of HZH. The treatment by 0.5% HZH in supercritical CO 2 (8 MPa, 35°C) for 30 min provided 58.3% of positive mutation rate and an expected mutant strain with about 76.7% increase in lipase yield compared with the wild strain. Possible mutagenesis mechanisms were further explored. The analysis on pH drop of the treated seed liquid was performed to better understand the interaction. Results proved that the induced mutation with enhanced yield of lipase could be achieved by co-mutagenesis of supercritical CO 2 and HZH.

Key words: Supercritical CO 2, Hydrazine hydrate, Mutation, pH Drop, Lipase, Flavobacterium sp.

INTRODUCTION applications. It is known that stress is a disturbance to the normal functioning of a Lipases are well-known enzymes extensively used biological system and mutagenesis is often in industrial biotransformation processes, which increased in the bacterial populations as a usually exhibit a good chemioselectivity, consequence of stress-induced genetic pathways regioselectivity, enantioselectivity and possess a (Tenaillon et al. 2004; Foster 2005). A previous broad substrate specificity exhibiting optimum study had demonstrated that supercritical CO 2 was activities over a wide range of temperatures a promising environmentally-friendly mutagen, or (Bertoldo et al. 2011). Flavobacterium sp. strain co-mutagen owing to its distinctive stress (Zhang YY25 produced an intermediate temperature et al. 2008). alkaline lipase, which had stable and higher Carbon dioxide (CO 2) is an inert, inexpensive, enzyme activity in tert-butanol, a common organic easily available, odorless, tasteless, environment- medium. Fermentation for 24 h provided the strain friendly, and GRAS (Generally Recognized As a maximum level of lipase yield, about 16.4 u/mL. Safe) solvent. The supercritical technology is a Mutations of strains may be induced by the green sustainable process in which the solvents’ treatment with physical, or chemical mutagens to power and selectivity can be relatively easy to tune improve lipase yield for the sake of better according to the operating conditions (Hegel et al.

*Author for correspondence: [email protected]

Braz. Arch. Biol. Technol. v.56 n.4: pp. 541-546, July/Aug 2013 542 Zhang, Q. et al.

2011). CO 2 is frequently used in near-, or stored in refrigerator on agar slant (beef extract supercritical conditions, wherein it shows 0.5%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.5). excellent mass transfer properties in terms of In order to prepare the seed liquid, a loop of cells penetration into matrices (Valverde et al. 2010). from the slant was inoculated into 50 mL of seed Possibly due to assist in the permeability, medium (beef extract 0.5%, peptone 1%, NaCl supercritical CO 2 had been found to enhance the 0.5%, pH 7.5) and cultivated with shaking at 30°C, sterilization effects and/or mutagenicity of the 200 rpm for 16h, which contained a cell additives (co-solvents) such as hydrogen peroxide concentration of about 10 8 cfu/mL. For the (H 2O2) (Zhang et al. 2006), dimethylsulfoxide production of lipase, 5.0 mL of seed liquid was (DMSO) (Zhang et al. 2008), etc. The solvent inoculated into 50 mL of fermentation medium properties of supercritical CO 2 could also be (peptone 0.5%, yeast extract 0.2%, beef extract drastically changed by the addition of a small 0.3%, K 2HPO 4 0.1%, NaCl 0.1%, MgSO 4 0.01%, amount of polar solvent as a modifier (Sicardi et olive oil 1%, glycerol 0.5%, Triton X-100 0.1%, al. 2000). pH 7.0) and cultivated with shaking at 30°C, 200 Hydrazine derivatives are widely used compounds rpm for 24 h. in the pharmaceutical, agrochemical, polymer and dye industries, and also as precursors in organic Mutation treatment synthesis. Many of them show significant Various doses of HZH were added independently biological activity and several are shown to be into the seed liquid and shaken up before effective for the treatment of tuberculosis, supercritical treatment. HZH (0.5 mL at 64 wt.% Parkinson’s disease and (Bredihhin hydrazine) in 50 mL of seed liquid expressed 1.0% and Mäeorg 2008). Some hydrazine compounds, content. A batch procedure was used for the hydrazine hydrate (HZH), (PHZ), treatment of the prepared seed liquid by hydralazine (HLZ) and (NLD), have supercritical CO 2. Figure 1 represented the scheme been reported mutagenic in strain WP2 of the apparatus. After the system was cleaned uvrA /pKM101. HZH could induce a higher level with 75% (v/v) aqueous solution and then of revertants, which carried suppressor mutations sterile distilled water to prevent possible resulting exclusively from G:C–A:T transitions contamination, 50 mL of seed liquid with HZH (Blanco et al. 1998). was loaded into the reactor with thermostat and the In order to obtain a mutation with enhanced yield pipelines were linked tightly. Then CO 2 was of lipase, here HZH was employed to treat pumped into the reactor with the high-pressure Flavobacterium sp. strain YY25 through adding plunger-pump. The pressure and temperature were straight into the seed liquid before treatment by held for 30 min after the system reached 8 MPa, supercritical CO 2. The co-effects of supercritical 35°C. Afterwards, gas-valve was opened to let out CO 2 and HZH on the survival rate and positive the CO 2 until a complete discharge of pressure was mutation rate of the tested strain were examined, attained. The samples from different runs of and possible action mechanisms were explored in treatments were taken out from the reactor. The this work. control sample without HZH received the same treatment as the treated sample except that it was kept at atmospheric pressure. MATERIALS AND METHODS Cell viability measurement Strains and culture conditions In order to explore the survival kinetics, the serial Flavobacterium sp. strain YY25, a wild bacterium dilution-agar pour plate procedure was employed that produced extracellular lipase, was used as a to quantify viable cells in 50 mL of seed liquid. tested strain. Using a selective medium, it was The treated seed liquid as well as the control was isolated from the fat soil sample around piggery. diluted serially with the sterile water by a factor of The strain was a Gram-negative, rod-shaped ten until a moderate dilution was attained. After bacterium and showed yellow circular colonies on 1.0 mL of the moderate dilution was added into the agar plate. It was identified Flavobacterium the Petri-dish, about 15 mL of agar medium (beef based on its physiological and biochemical extract 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, characteristics using automatic microorganism pH 7.5) was poured promptly. They were mixed identification instrument. The tested strain was and incubated for 48 h at 30°C. The count of

Braz. Arch. Biol. Technol. v.56 n.4: pp. 541-546, July/Aug 2013 Supercritical CO 2 and Hydrazine Hydrate 543

different seed liquids was repeated in parallel for the arithmetic means of Log ( N/No ) of minimum three times. The number of viable cells was three runs, where No was the number of cells in expressed as cfu/mL (cfu: colony forming unit). the control sample and N was the number of cells The values of survival rate were calculated with in the treated sample.

P T CO 2 3

P 5

1 2

4

Figure 1 - Supercritical CO 2 treatment system. 1, CO 2 cylinder; 2, filter; 3, Cooling bath; 4, high- pressure plunger-pump; 5, reactor (thermostat of the pressure vessel). The labels T and P refer to the temperature probe and the pressure gauge, respectively.

Cell mutation measurement obtained remained stable for at least 2 h. The After the treated seed liquid was poured into plate assayed mixture consisting of 2.95 mL of substrate and cultivated for 48 h for the count, single solution and 0.05 mL of suitably diluted enzyme colonies appeared on the agar medium plate. All was incubated at 50°C for 10 min. The pNPP the colonies from the plate of moderate colonies- released was measured at 410 nm in number were selected individually and inoculated spectrophotometer. One unit of lipase activity was into the slant medium (beef extract 0.5%, peptone defined as the amount of enzyme, which released 1%, NaCl 0.5%, agar 2%, pH 7.5). After 48 h of 1.0 µmoL of pNPP in 1 min under the above assay culture at 30°C, they were stored at 4°C as conditions. The lipase yield of the fermentation potential mutant strains. The selected potential liquid was expressed as u/mL. mutant strains as well as the wild strain were tested for lipase yield by fermentation. The Statistics positive mutation rate for each treatment was All the experiments were repeated for three to four calculated as the number of the positive mutant times. The data shown in the corresponding strains divided by the number of all selected figures were the mean values of the experiments, strains on the corresponding agar medium plate. and it was indicated that the relative standard deviations were all within ±5%. Lipase assays At the end of fermentation, the fermentation liquid was centrifuged at 6000 g for 15 min and the clear RESULTS AND DISCUSSION supernatant containing extracellular lipase was obtained. The spectrophotometric method was In a previous work, 8 MPa, 35°C and 30 min was used for rapid and routine measurement of lipase obtained as the acceptable treatment conditions of activity using pnitrophenylpalmitate (pNPP) as the supercritical CO 2 to obtain ideal mutant strains. substrate (Mahadik et al. 2002). The substrate The mutagenicity could be intensified by adding solution was prepared by adding 0.1 mL of 1.0% DMSO into the seed liquid before solution A (150 mg of pNPP in 50 mL of supercritical treatment. The corresponding positive isopropanol) into 2.85 mL of solution B (2.22 g of mutation rate was 45.6%. A mutant strain with Triton X-100 and 0.56 g of gum arabic in 500 mL about 44.2% increase in lipase yield than the wild of phosphate buffer, pH 9.0). The emulsion

Braz. Arch. Biol. Technol. v.56 n.4: pp. 541-546, July/Aug 2013 544 Zhang, Q. et al.

strain was obtained by supercritical CO 2 and 1.0% CO 2 in the air. Apparently, in the supercritical DMSO (Zhang et al. 2008). In order to obtain a system, higher concentration of HZH was easy to mutant strain with more productive lipase, a better react with CO 2 into a hydrazine compound, which substitute for DMSO was considered to be reduced the inactivation effects. explored. Results showed that 0.5 and 1.0% HZH treatments had no significant differences in the positive The effects of HZH on mutation in supercritical mutation rates, adding up to the larger values of treatment 58.3 and 59.6%, respectively. Pure supercritical HZH is a monohydrate of hydrazine and relatively CO 2 without HZH only provided the positive safer than anhydrous hydrazine (Satyanarayana mutation rates of 27.3%. Obviously, the mutation and Bhattacharya 2004). Here, HZH was used as a effects were strengthened greatly by 0.5%, or potential mutagen to treat the seed liquid in 1.0% HZH in supercritical treatment. More high- supercritical CO 2 (condition: 8 MPa, 35°C, 30 quality mutant strains and a selected mutant strain min). The results in Figure 2 showed that the of maximum lipase yield, with approximately effects of HZH in supercritical treatment on cell 76.7% increase than the wild strain, were obtained were content-related. The survival of the cells with 0.5% HZH treatment (Fig. 3). It suggested decreased with the increase in HZH content and that 0.5% HZH was a better mutation dose than reached a minimum at 1.0%. Afterwards, the 1.0% HZH. survival rate slightly increased. HZH can absorb

-2.0 70

-2.5 60 50

) -3.0 No

/ 40

N -3.5 30 Log( -4.0 20

-4.5 10 Positivemutation rate (%)

-5.0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0

HZH (%)

Figure 2 - Effects of HZH in supercritical CO2 on survival rate (Log (N/No)) and positive mutation rate. Legend: ■, the survival rate; □, the positive mutation rate. Conditions: pressure, 8 MPa; temperature, 35 °C; treatment time, 30 min; HZH, variable.

180 170 160 150 140 130 120 Relative lipase yield (%) yield lipase Relative 110 100 0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728 Positive mutant strain number

Figure 3 - Relative lipase yield of the selected positive mutant strains after 0.5% HZH and 1.0% HZH treatments, respectively. Legend: □, 0.5% HZH treatment; ▲, 1.0% HZH treatment. Conditions: pressure, 8 MPa; temperature, 35 °C; treatment time, 30 min; HZH, variable.

Braz. Arch. Biol. Technol. v.56 n.4: pp. 541-546, July/Aug 2013 Supercritical CO 2 and Hydrazine Hydrate 545

Although 0.5% HZH alone resulted in by direct mis-pairing at replication rather than by mutagenicity, it brought lower positive mutation error-prone repair (Kimball 1977). Here, five rate (only 8.3%) than the case of HZH in successive cultures of the selected mutant strain supercritical CO 2. Perhaps supercritical CO 2 were further performed under the same facilitated transient permeability and induced fermentation conditions. All the cultures showed mutation function of the additive (Zhang et al. almost the same lipase yield, indicating the 2008). Other authors have demonstrated that the stability of the mutant genotype. activity of some intracellular enzymes declined, or lost after supercritical treatment such as ATPase, The effects of HZH on pH drop in the seed phosphatase, peroxidase, etc (Bertoloni et al. 2006; liquid Gui et al. 2006; Liu et al. 2008). So it was also During the treatment of microorganisms by presumed that certain relevant enzymes with lipase supercritical CO 2 in an aqueous medium, the production might be induced, or changed by dissolution of CO 2 into the liquid phase is a natural supercritical CO 2 and HZH. process that leads to a pH decrease in the medium. HZH was proved an alternative additive of DMSO Therefore, pH drop had been considered to be one in the mutation treatment by supercritical CO 2 due key in the supercritical inactivation of to its more powerful mutagenicity. The chemical microorganisms (Kim et al. 2007; Bortoluzzi et al. properties of HZH derive from hydrazine 2011). Here, a pH meter was employed to measure (Satyanarayana and Bhattacharya 2004). the pH value of the seed liquid before and after the Hydrazine can react with the pyrimidines in DNA treatment by HZH and supercritical CO 2. The to form N4-aminocytosine. The amination of results of pH drop (Fig. 4) showed three stages: cytosine may be mutagenic for bacteria. The the stable first stage, the second stage of a rapid mutations from hydrazine are probably produced fall, and the third stage of a slight rise.

1.8 1.7 1.6 1.5 1.4

pHDrop 1.3 1.2 1.1 1.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0

HZH (%)

Figure 4 - Effects of HZH in supercritical CO2 on pH drop of the seed liquid. Conditions: pressure, 8 MPa; temperature, 35 °C; treatment time, 30 min; HZH, variable.

HZH is a polar solvent and can offer an alkaline of HZH (0.5%) had hardly any effect on the environment for the seed liquid because of the medium environment. Apparently the mutagenic highly alkaline nature of hydrazine (Satyanarayana function was derived from the generated and Bhattacharya 2004). The 0.5% HZH alone compound of HZH and CO 2. resulted increase in the pH in the seed liquid (data Supercritical CO 2 can cause the chemical not shown). But 0.5% HZH in the supercritical modification of the lipid double-layer of cell CO 2 had almost the same pH drop as without membrane, thus increasing dramatically its HZH. The change of pH drop reflected a balance permeability, which contributes to interaction with between the dissolution of CO 2 and the reaction of the intracellular materials (Spilimbergo et al. HZH with CO 2 in an aqueous medium. Low dose 2007). Higher doses of HZH (1.0%, or above) had

Braz. Arch. Biol. Technol. v.56 n.4: pp. 541-546, July/Aug 2013 546 Zhang, Q. et al.

less pH drop than pure supercritical CO 2. They Gui F, Chen F, Wu J, Wang Z, Liao X, Hu X. produced more hydrazine molecules to neutralize Inactivation and structural change of horseradish peroxidase treated with supercritical carbon dioxide. the acidification of CO 2. It was presumed that HZH surrounding the cells affected the action of Food Chem. 2006; 97: 480-489. supercritical CO and then the potential Hegel PE, Camy S, Destrac P, Condoret JS. Influence 2 of pretreatments for extraction of lipids from yeast by mutagenesis path from the modification of using supercritical carbon dioxide and ethanol as membrane was blocked, which reduced the cosolvent. J Supercrit Fluid. 2011; 58: 68-78. mutation effects. Kim SR, Rhee MS, Kim BC, Lee H, Kim KH. Modeling of the inactivation of Salmonella typhimurium by supercritical carbon dioxide in CONCLUSIONS physiological saline and phosphate-buffered saline. J Microbiol Meth. 2007; 70: 132-141. The effects of HZH in supercritical treatment on Kimball RF. The mutagenicity of hydrazine and some the survival rate and positive mutation rate of the of its derivatives. Mutat Res. 1977; 39: 111-126. tested strain were related to its concentration. The Liu X, Gao Y, Peng X, Yang B, Xu H, Zhao J. analysis on pH drop of the treated seed liquid Inactivation of peroxidase and polyphenol oxidase in red beet ( Beta vulgaris L.) extract with high pressure provided some possible explanations for the co- carbon dioxide. Innov Food Sci Emerg. 2008; 9: 24- mutagenesis mechanisms. An expected positive 31. mutant strain with higher lipase yield (about 29.0 Mahadik ND, Puntambekar US, Bastawde KB, Khire u/mL) was obtained by the synergic action of HZH JM, Gokhale DV. Production of acidic lipase by (0.5%) and supercritical CO 2 (8 MPa, 35°C) on the Aspergillus niger in solid state Fermentation. Process seed liquid for 30 min. As far as mutation was Biochem. 2002; 38: 715-721. concerned, HZH was a promising additive in Satyanarayana SV, Bhattacharya PK. Pervaporation of hydrazine hydrate: separation characteristics of supercritical treatment and supercritical CO 2 was a potential co-mutagen as well. membranes with hydrophilic to hydrophobic behaviour. J Membrane Sci. 2004; 238: 103-115.

Sicardi S, Manna L, Banchero M. Diffusion of disperse dyes in PET films during impregnation with a REFERENCES supercritical fluid. J Supercrit Fluid. 2000; 17: 187- 194. Bertoldo JB, Razzera G, Vernal J, Brod FCA, Arisi Spilimbergo S, Mantoan D, Dalser A. Supercritical ACM, Terenzi H. Structural stability of gases pasteurization of apple juice. J Supercrit Fluid. Staphylococcus xylosus lipase is modulated by Zn 2+ 2007; 40: 485-489. ions. Biochim Biophys Acta. 2011; 1814: 1120-1126. Tenaillon O, Denamur E, Matic I. Evolutionary Bertoloni G, Bertucco A, Cian DV, Parton T. A study significance of stress-induced mutagenesis in on the inactivation of micro-organisms and enzymes bacteria-a review. Trends Microbiol. 2004; 12: 264- by high pressure CO 2. Biotechnol Bioeng. 2006; 95: 270. 155-160. Valverde MT, Marín-Iniesta F, Calvo L. Inactivation of Blanco M, Martínez A, Urios A, Herrera G, O’Connor Saccharomyces cerevisiae in conference pear with JE. Detection of oxidative mutagenesis by high pressure carbon dioxide and effects on pear and other hydrazine derivatives in Escherichia coli quality. J Food Eng. 2010; 98: 421-428. WP2 tester strain IC203, deficient in OxyR: strong Zhang J, Burrows S, Gleason C, Matthews MA, Drews protective effects of rat S9. Mutat Res. 1998; MJ, LaBerge M, et al. Sterilizing Bacillus pumilus 417: 39-46. spores using supercritical carbon dioxide. J Microbiol Bortoluzzi D, Cinquemani C, Torresani E, Spilimbergo Meth. 2006; 66: 479-485. S. Pressure-induced pH changes in aqueous solutions Zhang QY, Qian JQ, Guo H, Yang SL. Supercritical - On-line measurement and semi-empirical modelling CO 2: A novel environmentally friendly mutagen. J approach. J Supercrit Fluid. 2011; 56: 6-13. Microbiol Meth. 2008; 75: 25-28. Bredihhin A, Mäeorg U. Effective strategy for the systematic synthesis of hydrazine derivatives. Tetrahedron. 2008; 64: 6788-6793. Received: June 28, 2012; Foster PL. Stress responses and genetic variation in Accepted: May 16, 2013. bacteria-a review. Mutat Res. 2005; 569: 3-11.

Braz. Arch. Biol. Technol. v.56 n.4: pp. 541-546, July/Aug 2013