Research Article Plackett-Burman Design for Rgilcc1 Laccase Activity Enhancement in Pichia Pastoris: Concentrated Enzyme Kinetic Characterization
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Hindawi Enzyme Research Volume 2017, Article ID 5947581, 10 pages https://doi.org/10.1155/2017/5947581 Research Article Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization Edwin D. Morales-Álvarez,1,2 Claudia M. Rivera-Hoyos,3 Ángela M. Cardozo-Bernal,3 Raúl A. Poutou-Piñales,3 Aura M. Pedroza-Rodríguez,1 Dennis J. Díaz-Rincón,4 Alexander Rodríguez-López,4 Carlos J. Alméciga-Díaz,4 and Claudia L. Cuervo-Patiño5 1 Laboratorio de Microbiolog´ıa Ambiental y de Suelos, Grupo de Biotecnolog´ıa Ambiental e Industrial (GBAI), Departamento de Microbiolog´ıa, Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogota,´ Colombia 2Departamento de Qu´ımica, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Caldas, Colombia 3Laboratorio de Biotecnolog´ıa Molecular, Grupo de Biotecnolog´ıa Ambiental e Industrial (GBAI), Departamento de Microbiolog´ıa, Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogota,´ Colombia 4Laboratorio de Expresion´ de Prote´ınas, Instituto de Errores Innatos del Metabolismo (IEIM), Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogota,´ Colombia 5Laboratorio de Parasitolog´ıa Molecular, Grupo de Enfermedades Infecciosas, Facultad de Ciencias, Pontificia Universidad Javeriana (PUJ), Bogota,´ Colombia Correspondence should be addressed to Raul´ A. Poutou-Pinales;˜ [email protected] Received 11 January 2017; Revised 27 February 2017; Accepted 9 March 2017; Published 21 March 2017 Academic Editor: Hartmut Kuhn Copyright © 2017 Edwin D. Morales-Alvarez´ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design −1 allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL at 168 h of culture in a ∘ 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50 C and retained a residual enzymatic activity greater ∘ ∘ than 70% at 60 Cand50%at70C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activitygreaterthan90%.Thelowestresidualactivitygreaterthan55%wasobtainedatpH10.0± 0.2. Furthermore, calculated −5 −1 −2 apparent enzyme kinetic parameters were a max of 6.87 × 10 mM s ,withanapparent of 5.36 × 10 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges. 1. Introduction is oxidized by donating its electron, where molecular oxygen acts as an electron acceptor and is reduced into water [1–3]. Laccases are blue multicopper oxidases (EC 1.10.3.2), cat- Laccases are enzymes mainly produced in white rot alyzing oxidation reactions for an array of compounds such fungi; however they are widely distributed in plants, insects, as diphenols, polyphenols, deamines, aromatic amines, inor- fungi, and bacteria [1, 4–6]. They have important applications ganic compounds, and nonphenolic compounds in the pres- in different industrial settings, as they help to reduce the ence of redox mediators. During the reaction the substrate environmental impact of their waste. These applications 2 Enzyme Research includedyebleachingintextileindustry,bleachingofcellu- 1.0 mM), inoculum percentage (2 and 10% (v/v)), glucose −1 lose pulp, detoxification of residual waters, toxic compound concentration (10 and 30 gL ), NH4SO4 concentration (5 −1 bioremediation, biosensor construction, fuel cells, fruit juice and 20 mM), peptone concentration (10 and 20 gL ), and −1 processing, and synthesis of molecules in the pharmaceutical yeast extract concentration (5 and 10 gL ). The design industry [3, 6–10]. included a central point evaluated three times; values within Laccase biotechnological and environmental applica- central points were 225 mL media, 0.55 mM CuSO4,6% −1 −1 tions require great enzyme quantities; unfortunately laccases inoculum (v/v), 20 gL glucose, 12.5 mM NH4SO4,15gL −1 obtained from natural sources are not suitable for long growth peptone, and 7.5 gL yeast extract [22]. For statistical analysis periods, low product/biomass (/) or product/substrate −1 theresponsevariableevaluatedwasenzymeactivity(UL ). (/) yield, and prolonged, complex, and costly isolation Additionally, to determine if statistically significant differ- procedures [3, 6, 11]. Therefore, heterologous expression is ences were observed among treatments (1–12)aone-way a promising option for greater scale production, using the ANOVA with Tukey post hoc test was performed, employing potential of hosts that are easy to handle and culture, such a 95% confidence interval (CI, = 0.05). Moreover, Shapiro- as bacteria and yeast [12]. Wilk normalization test was applied to verify data quality Yeast offers fast growth rates, ease of gene manipulation, using SAS V 9.05 2004 (SAS Institute Inc., Cary, NC, USA). and posttranslational modification capabilities. P. p a stor i s has −1 been employed for years as an industrial platform for het- Note.Enzymeactivity(UL ), protein concentration (mg erologous protein expression. Moreover, it is one of the most −1 −1 mL ), and glucose concentration (gL ), specific activity effective expression systems to obtain high yield extracellular −1 −1 −1 −1 proteins [14–17]. Additionally, various reports have described (UL mg mL ), and productivity (UL h )wereassayed better expression and productivity levels thorough culture 0 to 12 h every two hours. The same variables were then media optimization [4, 18]. evaluatedevery24huptotheend(168h),sincepreliminary data revealed that better results were obtained between 156 h The objective of this work was to increase Ganoderma −1 lucidum rGILCC1 laccase activity in Pichia pastoris at low and 168 h [19]. Enzyme activity (UL )wastheresponse variable utilized for statistical analyses. All improvement scale in the laboratory to determine pH and temperature ∘ assays were carried out in 500 mL Erlenmeyer flasks at 30 C stability and define its max and in concentrated super- and 180 rpm, employing the same flask for the total 168 hof natant obtained from microbial culture. This was achieved ± by improving the following factors: nitrogen and source culture at variable pH starting at 7 0.2. Design Expert V.9.0 type (organic and inorganic), carbon concentration (glu- (Stat-Ease, Inc., Minneapolis, MN, USA) software was used cose), copper concentration, oxygen transfer (media vol- to devise experimental design and result analysis. In addition, ume/Erlenmeyer flask volume ratio), time of culture, and Sigma Plot V.11.0 software (Systat Software Inc. Sane, Jos´ CA, inoculum percentage. USA) was employed to graph concentrated enzyme kinetics and results. 2. Materials and Methods 2.4. Supernatant Concentrate. Culture supernatant demons- 2.1. Strain. The strain was P. p a stor i s X33 containing pGA- trating the highest enzymatic activity values with ABTS PZA-LacGluc-Stop (Clone 1) expression vector with previ- substrate [2,20-azino-bis(3-ethylbenzothiazoline-6-sulpho- ously optimized synthetic gene GILCC1 coding for Gano- nic acid)] under conditions previously described [23] (17.9 derma lucidum GILCC1 laccase. This strain was kept in 1% −1 −1 mg mL protein and 4.69 ± 0.39 UL enzyme activity, with YPD (w/v), 2% peptone, 1% yeast extract, and 2% D+ glucose, −1 − ∘ a specific enzyme activity of 0.26 Umg at 168 h of culture) supplemented with 20% glycerol (w/v), and kept at 80 C ∘ [19–21]. was used. Briefly, culture was centrifuged at4 C 8,000 and supernatant was filtered in a serial manner through What- manNumber1filterpaper,followedby0.45and0.22m 2.2. Inoculum Preparation. Pichia pastoris X33/pGAPZ A- membranes (Pall Corp, Port Washington, NY, USA). The LaccGluc-Stop clone 1 Master Cell Bank (MCB) [21] was filtrate was concentrated by centrifugation employing a thawedandusedforinoculating5mLscrewcaptubes −1 10 kDa Ultracel regenerated cellulose membrane (Millipore, with sterile YPD supplemented with 40 gmL zeocin ∘ Billerica, MA, USA) [23]. Approximately 20 mL concentrate (Z). Tubes were incubated overnight (ON) at 30 Cwith was used to perform the enzyme’s functional identity by 180 rpm agitation, followed by inoculation under the same zymography. conditions for 12 h in 500 mL Erlenmeyer flasks, containing 100 mL (effective work volume) of fresh YPD-Z. The resulting culture was verified by Gram stain to detect presence of 2.5. Enzyme Functional Identification. Zymogram was ran contaminating morphologies and used for factorial design in 12% (w/v) native PAGE under nondenaturing conditions. inoculations. Activity or functionality was visualized by 0.5 M ABTS stain. BenchMark6 Pre-Stained Protein ladder (Life Technolo- 2.3. Plackett-Burman Experimental Design (PBED). Seven gies6, USA) was used as the molecular weight standard factorswereevaluatedwithtwolevelseachasfollows:media