Recombinant Rhizopus Oryzae Lipase Research Articles

Second- and third-generation biodiesel production with immobilised recombinant Rhizopus oryzae lipase: Influence of the support, substrate acidity and bioprocess scale-up

Rhizopus oryzae lipase immobilised onto differently functionalised polymethacrylate (Purolite®) and magnetite superparamagnetic supports was assessed as a catalyst for biodiesel production with pomace oil. The presence of surface hydrocarbon chains increased the operational stability of the biocatalysts supported on Purolite® and superparamagnetic particles up to 9 and 2 times, respectively. By contrast, the presence of functional groups had no effect on the initial transesterification rate, which was twice higher with the lipase immobilised onto Purolite®. Also, functionalising Purolite® with epoxide and octadecyl groups led to the highest biodiesel and volumetric productivity. This biocatalyst with other substrates including makauba, jatropha, waste cooking oil, and microbial oil, led to similar initial reaction rates. However, simply raising substrate acidity from 0.5 to 2% increased the operational stability of the biocatalysts 15 times. A synergistic effect between acyl-acceptor concentration and substrate acidity was observed. The transesterification reaction was successfully scaled up to 50 mL.

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

Recombinant Rhizopus oryzae lipase (mature sequence, rROL) was modified by adding to its N-terminal 28 additional amino acids from the C-terminal of the prosequence (proROL) to obtain a biocatalyst more suitable for the biodiesel industry. Both enzymes were expressed in Pichia pastoris and compared in terms of production bioprocess parameters, biochemical properties, and stability. Growth kinetics, production, and yields were better for proROL harboring strain than rROL one in batch cultures. When different fed-batch strategies were applied, lipase production and volumetric productivity of proROL-strain were always higher (5.4 and 4.4-fold, respectively) in the best case. rROL and proROL enzymatic activity was dependent on ionic strength and peaked in 200 mM Tris-HCl buffer. The optimum temperature and pH for rROL were influenced by ionic strength, but those for proROL were not. The presence of these amino acids altered lipase substrate specificity and increased proROL stability when different temperature, pH, and methanol/ethanol concentrations were employed. The 28 amino acids were found to be preferably removed by proteases, leading to the transformation of proROL into rROL. Nevertheless, the truncated prosequence enhanced Rhizopus oryzae lipase heterologous production and stability, making it more appropriate as industrial biocatalyst.

Towards optimal substrate feeding for heterologous protein production in Pichia pastoris (Komagataella spp) fed‐batch processes under PAOX1 control: a modeling aided approach

AbstractBACKGROUNDTo improve the efficiency of a bioprocess, key parameters, such as yield, titer and productivity, must be considered. They are mainly dependent on the specific rates for product (qP) and cell growth (μ), and their correlation determines the most suitable feeding polices that should be applied.RESULTSThe mathematical description of the Pichia pastoris (Komagataella spp) PAOX1‐based system (Mut+) expressing recombinant Rhizopus oryzae lipase (ROL), which includes cell growth, substrate consumption and product formation kinetics, was used to define the optimal profiles regarding methanol feeding‐rate (F), residual methanol concentration (S) and specific growth rate (μ). Optimal trajectories obtained using numerical optimization and algorithms based on S‐profile determination were first applied to manage the multiplicity of S‐states with both μ and qP. Once the S‐profile was determined, the F and μ profiles were obtained in a straightforward manner from mass balances and growth kinetics. From experimental results, the bioprocess efficiency improvement was confirmed in terms of a 2.2‐fold higher final titer and 3.4‐fold higher productivity compared with the best standard pre‐programmed exponential substrate feeding strategy. Additionally, 1.2‐fold higher productivity and 1.1‐fold higher final titer relative to the most promising feedback control of the methanol concentration strategy were obtained.CONCLUSIONThe whole model‐based approach was established as a versatile and simple platform, which would then be transferable to alternative modes of operation and even to produce other proteins of interest. © 2018 Society of Chemical Industry

Effect of acyl‐acceptor stepwise addition strategy using alperujo oil as a substrate in enzymatic biodiesel synthesis

AbstractBACKGROUNDUsing renewable feedstock sources for biodiesel production seems to be a promising strategy and even more so when enzymatic catalysis with lipases are used. However, it is well known that these enzymes could be inactivated by reaction conditions such as temperature or alcohol concentration. In this work, the effect of temperature and initial water activity (aw) value on immobilised recombinant Rhizopus oryzae lipase (rROL) were studied. Methanolysis and ethanolysis reactions using alperujo oil with three different stepwise addition strategies were employed.RESULTSRecombinant 1,3‐positional selective rROL covalently immobilised on polymethacrylate amino‐epoxy activated support showed maximum initial reaction rate at low aw value (0.093). It was found that 30 °C was the optimal temperature in terms of biocatalyst stability during transesterification reactions. Adding alcohol at once, ethanol was clearly a better acyl‐acceptor in terms of stability than methanol. Productivity was found to be 2‐fold higher when five pulses of ethanol were used instead of methanol.CONCLUSIONSAlperujo oil has great potential as a low cost feedstock for biodiesel production through enzymatic catalysis using a nearly semi‐continuous alcohol addition strategy. © 2017 Society of Chemical Industry

Exploring substrate specificities of a recombinant Rhizopus oryzae lipase in biodiesel synthesis

The alcoholysis of triolein was used to explore the specific features of a recombinant Rhizopus oryzae lipase (rROL) for biodiesel synthesis. For this purpose, different acylglycerols were compared as substrates in lipase-catalysed transesterification. rROL was shown to exhibit a higher specificity towards 1-monoolein than triolein compared to other R. oryzae lipases, being more than 4-fold more specific; in contrast, rROL did not accept 2-monoolein as substrate, concluding that it is highly 1,3-positional specific. Comparing ethanol and methanol as acyl-acceptors, it was observed that the latter caused more lipase inactivation. Regarding alcohols, it was also demonstrated that acyl migration occurred in moderate alcohol concentrations.

Rice husk ash as a potential carrier for the immobilization of lipases applied in the enzymatic production of biodiesel

The potential application of rice husk ash (RH26) as support for the immobilization of a recombinant Rhizopus oryzae (rROL) lipase as biocatalyst in the enzymatic biodiesel production using alperujo oil and the comparison with commercial hydrophobic support OD403 (RelOD) has been made. Although the specific activity (UA mg support−1) was around one-half lower in RH26 than in RelOD when they were used as biocatalyst in biodiesel reaction, the normalized initial rate was similar, between 1.6 and 2.4 μmol FAME mL−1 mL−1 UA−1. Thus in terms of biocatalysis performance, rice husk as is an alternative to commercial supports. However, the main problem is the more complex recovery of RH26 for the reutilization compared with commercial ones.

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.

Improved ethyl butyrate synthesis catalyzed by an immobilized recombinant Rhizopus oryzae lipase: A comprehensive statistical study by production, reaction rate and yield analysis

The combined effect of the substrates on the ethyl butyrate (EB) synthesis (pineapple flavour) by a recombinant lipase from Rhizopus oryzae (rROL) immobilized in Octadecyl-Sepabeads has been studied. The initial concentration of butyric acid (BA) and the molar ratio butyric acid:ethanol (BA:ET) were selected as the key parameters to describe this combined effect. The study was focused on the maximization of the flavour production by applying a central composite rotatable experimental design which results were fitted to empirical models based on the response surface methodology. The obtained model predicts a major production of 0.118mmol/h at 54.6mM of BA initial concentration and at BA:ET initial molar ratio of 1.42. These results were validated through experimental biosynthesis reaction, obtaining a difference of less than 1%. Besides, in order to discuss in a more comprehensive approach the obtained production profile, the initial reaction rate and conversion profiles were also analyzed. This broad study shown that the highest initial reaction rate was predicted at 380mM of BA initial concentration and at 1.45 of BA:ET initial molar ratio. These conditions were different from those that lead to the highest yield since it was shown to be strongly dependent on the initial BA initial concentration but not significantly dependent on BA:ET initial molar ratio.

Enzymatic biodiesel synthesis from yeast oil using immobilized recombinant Rhizopus oryzae lipase

The recombinant Rhizopus oryzae lipase (1–3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

We have developed a yeast Pichia pastoris system for the high-level expression of recombinant Rhizopus oryzae lipase (ROL), which is a potentially effective catalyst in the solvent-free production of biodiesel fuel. In the glycerol fed-batch phase, the combination of the dissolved-oxygen-stat and gradient-control glycerol feeding strategies resulted in a higher cell biomass in the P. pastoris culture, with shorter feed times. In the methanol fed-batch phase, a constant methanol concentration of 0.3-0.5% (v/v) was found to be optimal for high ROL activity, maximum protein concentration, and a maximum biomass. Cells were grown at 30 °C and induced by methanol at 22 °C and a pH of 6.0 with the addition of 0.5% (w/v) casein, which reduced the proteolytic degradation of ROL. Using a combined glycerol step-increasing, on-line methanol feeding strategy and these reducing proteolytic degradation methods, a maximum biomass of 96 g DCW/l, a maximum concentration of 2.0 g/L, and an activity of 1302.2 U/mL ROL were attained. The simple culture process is the highest level of ROL expression reported in P. pastoris. Our results strongly demonstrate that the efficiency of the recombinant ROL expression is directly dependent on feed strategies, lower induction temperature, maintaining the proper pH, and the addition of casein.

Searching the best operational strategies for Rhizopus oryzae lipase production in Pichia pastoris Mut+ phenotype: Methanol limited or methanol non-limited fed-batch cultures?

Two different operational strategies, methanol limited (MLFB) and methanol non-limited fed-batch (MNLFB) cultures, have been compared for the production of the recombinant Rhizopus oryzae lipase (ROL) expressed in Pichia pastoris Mut+ phenotype. Yields, productivities and specific production rate in all MLFB conditions were very low, obtaining the best results at the lower specific growth rates. Similar results were obtained for MNLFB strategy at methanol set-point concentrations up to 2gl−1. However, for methanol set-points higher than 2gl−1 a significant increase in the production was observed. Methanol set-point of 3gl−1 was the optimum to maximize product yield (YP/X), both volumetric and specific productivity and mean specific production rate (qP,mean), although in terms of ROL production high values were obtained in the range of 3–10gl−1. An inhibitory effect of methanol on cell growth as well as slowness on ROL production during early induction phase was observed at concentration of 10gl−1.

Biosynthesis of ethyl butyrate by immobilized recombinant Rhizopus oryzae lipase expressed in Pichia pastoris

A recombinant Rhizopus oryzae lipase expressed in Pichia pastoris has been immobilized in three different kinds of supports: EP100, Eupergit®CM and Octadecyl-Sepabeads. These immobilized derivatives have been used to catalyze the synthesis of ethyl butyrate. The effect of different parameters on initial reaction rate and yield has been studied for each biocatalyst. The variables selected for the study were: temperature, water content, shaking speed, enzyme loading, type of solvent and substrates molar ratio. The results showed the EP100 derivative as the best choice in terms of initial rate and yield but, when the residual activity as well as the reutilization capacity was studied, Octadecyl-Sepabeads exhibits the highest stability, so it was finally chosen as the best biocatalyst.

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40°C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30min of incubation at 55°C.

The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system

A recombinant Rhizopus oryzae lipase producing Mut s Pichia pastoris strain was used as a model organism to study the effect of mixed substrates (glycerol and methanol) on the specific product productivity. Different fed-batch cultivations were performed under three constant specific growth rates (0.02, 0.05 and 0.1 h −1), maintaining a constant methanol concentration of 2 g l −1. At the lowest μ tested (0.02 h −1), the specific productivity was 1.23 and 1.61 fold higher and the specific methanol consumption rate ( q sMeOH) was 3 and 3.5 fold higher than values obtained when μ was 0.05 and 0.1 h −1, respectively. This implies a relation between the q sMeOH and the specific productivity, yielding higher specific productivities whenever the consumption of methanol is higher. Although glycerol was maintained under limiting conditions in all μ tested, when the relation between the μ Gly and μ MeOH was larger than 4, an important decrease on the maximal activity value was observed. Finally, a comparison under the same conditions using glycerol or sorbitol as co-substrates was also performed, obtaining better specific productivity when sorbitol was used. In addition, protease activity was detected when glycerol was used as co-substrate.

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32kDa. The maximum activity of recombinant lipase obtained from Muts recombinant was 90IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35°C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60–100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.

Immobilized recombinant Rhizopus oryzae lipase for the production of biodiesel in solvent free system

Different kinds of macroporous and anion resins were applied to immobilize recombinant Rhizopus oryzae lipase in the initial screening test. Anion exchange resin Amberlite IRA-93 was the most proper support to immobilize R. oryzae lipase by covalent binding technology. The transesterification reaction catalyzed by the immobilized lipase was investigated in a solvent free system. It was found that the anion resins used as the immobilized carriers per se contributed to the promotion of acyl migration in the methanolysis reactions and improved biodiesel yield by about 4–8%. The highest biodiesel yield of 90.5% was achieved under the optimum conditions (enzyme dosage 24 U/goil, methanol to oil molar ratio 4.8:1, water content 60% by weight of oil, temperature 37 °C). There was no obvious loss in immobilized lipase activity after being consecutively used for 7 cycles in the transesterification reactions.

Biochemical characterization and studies of adsorption and immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris

Biochemical characterization and studies of adsorption and immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris

Enantioselective transesterification using lipase-displaying yeast whole-cell biocatalyst.

An enantioselective transesterification in non-aqueous organic solvent was developed by utilizing a lipase-displaying yeast whole cell biocatalyst constructed in our previous study. As a model reaction, optical resolution of (RS)-1-phenylethanol, which serves as one of chiral building blocks, was carried out by enantioselective transesterification with vinyl acetate. Recombinant Rhizopus oryzae lipase displayed on the yeast cell surface retained its activity in hexane, heptane, cyclohexane and octane. The effective amount of whole-cell biocatalyst in the reaction mixture was 10 mg/ml solvent. In a reaction mixture incubated for 36 h with molecular sieves 4A, the concentration of (R)-1-phenylethyl acetate reached 39.8 mM (97.3% yield) with high enantiomeric excess (93.3%ee). In contrast, a reaction mixture incubated without molecular sieves 4A produced little (R)- and (S)-1-phenylethyl acetate. The results obtained in this study demonstrate the applicability of the lipase-displaying yeast whole cell biocatalyst to bioconversion processes in non-aqueous organic solvents.

Preparation of high activity yeast whole cell bioctalysts by optimization of intracellular production of recombinant Rhizopus oryzae lipase

Yeast whole cell biocatalysts, which intracellularly overproduced a recombinant lipase with a pro-sequence from Rhizopus oryzae IFO4697 (rProROL) were constructed, and the content of active lipase in Saccharomyces cerevisiae cells was maximized by optimizing the cultivation procedure. rProROL was overproduced intracellularly under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL) as the inducible system and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter as the constitutive expression system. Enhancement of expression level of ProROL gene at the initial cultivation phase inhibited rProROL accumulation in yeast cells both in GAPDH promoter system by using high glucose concentration at 30 °C and in UPR-ICL system by using non-fermentable carbon sources. The highest intracellular lipase activity of 350.6 IU/l was obtained in the inducible UPR-ICL system with an initial glucose concentration of 0.5% at 30 °C. To prepare the efficient whole cell biocatalyst by intracellular overproduction of lipase, utilization of inducible UPR-ICL and the optimization of cultivation conditions such as temperature, carbon source and its initial concentration are important.

Effect of the truncation of the C-terminal region of Kex2 endoprotease on processing of the recombinant Rhizopus oryzae lipase precursor in the co-expression system in yeast

The Saccharomyces cerevisiae Kex2p is a membrane-bound endoprotease and cleaves at a C-terminal site of Lys–Arg of the α-factor precursor in late Golgi compartment. The complete cleavage of a secreted recombinant Rhizopus oryzae lipase (ROL) precursor (rProROL) having an internal Lys–Arg has been examined by the co-expression of the KEX2 gene. The cleavage of rProROL was not sufficient. The co-expression system of the truncated gene encoding the soluble form of Kex2p (sKex2p) lacking C-terminal 201 amino acids containing the transmembrane domain (TMD) was constructed. sKex2p co-expressed was detected in the culture medium and the carboxyl site of Lys(-30)–Arg(-29) in the prosequence of rProROL was completely cleaved. These results revealed that on the process of secretion, including in the Golgi apparatus and secretion vesicles, attack of sKex2p to the produced protein occurred more efficiently than that of Kex2p. The cells having the co-expression system of the gene encoding sKex2p will be useful for production of foreign proteins designed to cleave the internal Lys–Arg site for their activation or for the application of the prepro-α-factor leader sequence.