Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT1) was cloned into the expression vector pPICZα A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (φ(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of φ(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R²=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4·10^–5 % biotin, 1 % methanol and 1 % glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.Gen za ekstracelularnu glicerol kinazu iz Saccharomyces cerevisiae (GUT1) kloniran je u ekspresijski vektor pPICZα A i integriran u genom metilotrofnog kvasca Pichia pastoris X-33. Prisutnost GUT1 potvrđena je PCR analizom. Izdvojena su četiri klona, u kojima je ispitana funkcionalnost rekombinantnog enzima. Jedan je od ispitanih klonova imao aktivnost glicerol kinaze od 0,32 U/mL i specifičnu aktivnost proteina od 0,0025 U/mg. Podloga za maksimalnu proizvodnju biomase na tresilici s pomoću rekombinantnog kvasca Pichia pastoris optimirana je uporabom glicerola, volumnog udjela od 2,31 %, kao izvora ugljika. Za optimiranje je upotrijebljena metoda odzivnih površina. U preliminarnim su ispitivanjima, primjenom Plackett-Burmanovog dizajna, određeni najvažniji čimbenici što utječu na proizvodnju biomase, a to su: volumni udio glicerola (φ(Gly)) i vrijeme uzgoja (t). Daljnji su eksperimenti provedeni radi optimiranja proizvodnje biomase, a pratili su centralno složeni dizajn kao funkciju volumnog udjela glicerola i vremena. Volumni je udio glicerola imao pozitivni linearni utjecaj na proizvodnju biomase. Vrijeme uzgoja je također bitno utjecalo (na razini linearno pozitivnih i kvadratnih zavisnosti) na proizvodnju biomase. Eksperimentalni su se podaci dobro uklapali u konveksnu funkciju koja opisuje polinom drugoga reda, u kojem je biomasa funkcija obaju faktora (R²=0,946). Prinos i specifična aktivnost glicerol kinaze ponajprije su ovisili o dodatku glicerola i metanola podlozi. Sastav optimirane podloge za proizvodnju enzima bio je: 1 % kvaščeva ekstrakta, 1 % peptona, 100 mM fosfatnog pufera (pH=6,0), 1,34 % podloge s kvascem i dušikom, 4·10^-5 % biotina, 1 % metanola i 1 % glicerola, pomoću kojih je dobivena aktivnost glicerol kinaze od 0,89 U/mL i koncentracija ukupnih proteina od 14,55 g/L u podlozi nakon 48 sati uzgoja
