Use of Vero cell line to verify the biodetoxification efficiency of castor bean waste

Ricin is a toxic protein present in castor bean seeds (Ricinus communis). A toxic residue named castor bean waste is generated during biodiesel production process, such as that developed by PETROBRAS (the national petroleum company of Brazil). Solid-state fermentation (SSF) was used to detoxify castor bean waste through the Penicillium simplicissimum growth. After 24 h of fungal growth, the ricin was no longer identified by Sephadex G-50 gel chromatography. In order to verify the biological activity of ricin after several treatment stages, an in vitro assay using Vero cell line was carried out. Through this methodology, it was verified that after 24 and 48 h of treatment, the cell culture showed slightly growth inhibition. The waste was completely detoxified only after 72 h of fungal growth. This fact shows that an in vitro assay is important to verify the real efficiency of detoxification. Moreover, a relationship between the fungal protease production and the waste detoxification was observed.     

Adding value to a toxic residue from the biodiesel industry: production of two distinct pool of lipases from <Emphasis Type="Italic">Penicillium simplicissimum</Emphasis> in castor bean waste

In countries with a strong agricultural base, such as Brazil, the generation of solid residues is very high. In some cases, these wastes present no utility due to their toxic and allergenic compounds, and so are an environmental concern. The castor bean (Ricinus communis) is a promising candidate for biodiesel production. From the biodiesel production process developed in the Petrobras Research Center using castor bean seeds, a toxic and alkaline waste is produced. The use of agroindustrial wastes in solid-state fermentation (SSF) is a very interesting alternative for obtaining enzymes at low cost. Therefore, in this work, castor bean waste was used, without any treatment, as a culture medium for fungal growth and lipase production. The fungus Penicillium simplicissimum was able to grow and produce an enzyme in this waste. In order to maximize the enzyme production, two sequential designs–Plackett-Burman (variable screening) followed by central composite rotatable design (CCRD)—were carried out, attaining a considerable increase in lipase production, reaching an activity of 155.0 U/g after 96 h of fermentation. The use of experimental design strategy was efficient, leading to an increase of 340% in the lipase production. Zymography showed the presence of different lipases in the crude extract. The partial characterization of such extract showed the occurrence of two lipase pools with distinct characteristics of pH and temperature of action: one group with optimal action at pH 6.5 and 45°C and another one at pH 9.0 and 25°C. These results demonstrate how to add value to a toxic and worthless residue through the production of lipases with distinct characteristics. This pool of enzymes, produced through a low cost methodology, can be applied in different areas of biotechnology.     

Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g⁻¹ at 18 h in the laboratory bioreactor to 113 U g⁻¹ at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g⁻¹ to 10.4 U g⁻¹. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

Increased production of FAEEs for biodiesel with lipase enhanced Saccharomyces cerevisiae

In this study, we expressed lipase 2 from Candida sp. 99-125 in Saccharomyces cerevisiae, and tried direct biodiesel production. Driven by 3-phosphoglycerate kinase promoter, Lip2 showed high expression level in cytoplasm. SDS-PAGE analysis confirmed the successful lipase expression with a 40 kDa molecular weight. The enzyme assay indicated that lipase 2 had a specific activity of 12.12 μmol/min/mg toward p-nitrophenyl palmitate. Gas chromatography showed that the main fatty acids of S. cerevisiae lipids were palmitoleic acid (31.79%) and oleic acid (29.84%). By three-step addition of 4% ethanol to culture broth, the yield of fatty acid ethyl esters by recombinant S. cerevisiae reached 11.4 mg/g dry cell weight. This work proposed a novel pathway for S. cerevisiae that could be applied for producing biodiesel directly.     

Lipase production and Penicillium simplicissimum morphology in solid-state and submerged fermentations

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.     

Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp. 99-125

In this paper, immobilized lipase catalyzed biodiesel production from lard was studied. Using Candida sp. 99-125, the effect of temperature, water content, enzyme amount, solvent and three-step methanolysis were investigated. The optimal conditions for processing 1 g of lard were: 0.2 g immobilized lipase, 8 ml n-hexane as solvent, 20% water based on the fat weight, temperature 40 °C, and three-step addition of methanol. As a result, the fatty acid methyl esters (FAMEs) yield was 87.4%. The lipase was proved to be stable when used repeatedly for 180 h.     

Cellulose ethanol production from waste newsprint by simultaneous saccharification and fermentation using Saccharomyces cerevisiae KNU5377

A thermotolerant ethanol-fermenting yeast, Saccharomyces cerevisiae KNU5377, isolated from a sludge of a local industrial complex stream in Korea, was evaluated for its capability for lignocellulosic ethanol production from waste newsprint in high temperature. In this fermentation, most of dry-defibrated waste newspaper was first saccharified at 50 °C for 108 h using a commercial cellulase and, then with the last addition of dry-defibrated newsprints to the pre-saccharified broth, simultaneous saccharification and fermentation (SSF) of 1.0 L of reaction mixture was carried out at 40 °C, slowly being dropped from 50 °C, for further 72 h in a 5 L fermentor by inoculating the overnight culture of KNU5377. The maximum production of 8.4% (v/v) ethanol was obtained when 250 g (w/v)/L of dry-defibrated waste newspaper was used for ethanol production by SSF. These results suggest that S. cerevisiae KNU5377 is very useful for cellulose ethanol production by the SSF system.     

Influence of selected host plants on biology of castor whitefly,Trialeurodes ricini (Hemiptera: Aleyrodidae)

The development time, immature survivorship, immature size, tertiary sex ratio, pre-oviposition period, fecundity, and preferences of the castor whitefly, Trialeurodes ricini on four host plant species (castor, eggplant, cotton and green bean) were evaluated under laboratory conditions. Development time from egg to adult emergence was the longest on cotton (33.3 days), the shortest on green bean (25.4 days), and intermediate on eggplant (28.5 days) and castor (28.3 days). The survival rate was the highest on castor (92.5%), followed by those on green bean (80.2%) and eggplant (73.8%), and the lowest on cotton (42.6%). The sex ratio was the highest on cotton (♀:♂ = 2.45:1.00), the lowest on eggplant (♀:♂ = 0.75:1.00), and intermediate on castor and green bean (♀:♂ = 1.04:1.00). T. ricini immatures and adults were generally larger when reared on castor and eggplant than on other plants. The net reproduction rate (R₀) was 57.656 females per female per generation, the generation time (T) was 35.9 days, the intrinsic rate of increase (r_m) was 0.1128 eggs per female per day, the gross reproduction rate (GRR) was 108.04 eggs per female per generation, the finite rate of increase (λ) was 1.1194 females per female per day, and the doubling time (DT) was 6.1149 days. In both no-choice and two-choice tests, T. ricini adults preferred castor for feeding and oviposition.     

Potential use of whole cell lipase from a newly isolated Aspergillus nomius for methanolysis of palm oil to biodiesel

A highly active whole cell lipase (WCL) for efficient methanolysis of palm oil (PO) to biodiesel (BD) was prepared by isolation, cultivation and immobilization of lipase producing fungi. Fungi were screened from soil and the best isolate (PDA-6) identified as Aspergillus nomius exhibited maximum WCL methanolysis activity (1.4 g h⁻¹ g⁻¹) when inexpensive waste cooking oil was used as carbon source. The maximum BD yield with PDA-6 WCL reached 95.3% after 40 h at a lipase load 10% (w/w) of PO and methanol to PO molar ratio 5:1. The immobilization of PDA-6 cells within biomass suspended particle (BSP) made of polyurethane foam improved the repeated use of WCL and the remaining activity after 10 cycles was 88.2%. The PDA-6 WCL was more active in methanolysis of PO to BD than most WCLs previously reported. The newly isolated A. nomius is not only potential for producing WCL but also utilizing waste cooking oil.     

Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum

An inexpensive self-made immobilized lipase from Penicillium expansum was shown to be an efficient biocatalyst for biodiesel production from waste oil with high acid value in organic solvent. It was revealed that water from the esterification of free fatty acids and methanol prohibited a high methyl ester yield. Adsorbents could effectively control the concentration of water in the reaction system, resulting in an improved methyl ester yield. Silica gel was proved to be the optimal adsorbent, affording a ME yield of 92.8% after 7 h. Moreover, the enzyme preparation displayed a higher stability in waste oil than in corn oil, with 68.4% of the original enzymatic activity retained after being reused for 10 batches.     

Highly soluble expression and molecular characterization of an organic solvent-stable and thermotolerant lipase originating from the metagenome

A novel organic solvent-stable and thermotolerant lipase gene (designated ostl28) was cloned from a metagenomic library and overexpressed in Escherichia coli BL21 (DE3) in soluble form. OSTL28 contained 262 amino acids with relative molecular mass 30.1 kDa and isoelectric point 9.7. The optimum pH and temperature of the OSTL28 were 7.5 and 60 °C, respectively. OSTL28 was stable in the pH range of 4.5–9.5 and at temperatures below 65 °C. The enzyme could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl laurate with the highest activity of 236 U/mg (54,000 U/L). The recombinant OSTL28 was highly resisted to organic solvents, especially glycerol and methanol. The metal ions, with the exception of Hg²⁺ and Ag⁺, did not have any influence on enzyme activity, whereas non-ionic surfactants and Al³⁺ slightly activated the enzyme. These features indicate that it is a potential biocatalyst for biodiesel production.     

Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system

Biocatalytic synthesis is a promising environmentally friendly process for the production of biodiesel, a sustainable alternative fuel from renewable plant resources. In order to develop an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs) were prepared from a commercial enzyme preparation from a recombinant Aspergillus strain expressing Thermomyces lanuginosus lipase and used for synthesis of biodiesel from palm olein by ethanolysis. Reaction parameters, including catalyst loading, temperature, and oil/alcohol molar ratio have been systematically optimized. Addition of tert-butanol was found to markedly increase the biocatalyst activity and stability resulting in improved product yield. Optimized reactions (20%, w/w PCMC-lipase to triacylglycerol and 1:4 fatty acid equivalence/ethanol molar ratio) led to the production of alkyl esters from palm olein at 89.9% yield on molar basis after incubation at 45 °C for 24 h in the presence of tert-butanol at a 1:1 molar ratio to triacylglycerol. Crude palm oil and palm fatty acid distillate were also efficiently converted to biodiesel with 82.1 and 75.5% yield, respectively, with continual dehydration by molecular sieving. Operational stability of PCMC-lipase could be improved by treatment with tert-butanol allowing recycling of the biocatalyst for at least 8 consecutive batches with only slight reduction in activity. This work thus shows a promising approach for biodiesel synthesis with microcrystalline lipase which could be further developed for cost-efficient industrial production of biodiesel.     

Ectoine improves yield of biodiesel catalyzed by immobilized lipase

To improve the production of biodiesel by enzymatic conversion of triglycerides in cottonseed oil, compatible solutes were added to the solvent-free methanolysis system to prevent competitive methanol inhibition on the immobilized lipase (Novozym^® 435). The results indicated that the addition of ectoine increased biodiesel synthesis using a three-step methanol addition process. The concentration of methyl ester (ME) reached a maximum of 95.0% in the presence of 1.1 mmol/l ectoine, an increase of 20.9% compared to that in the absence of ectoine. On the other hand, excess ectoine decreased the ME concentration. Ectoine was also shown to enhance reuse of the immobilized lipase, significantly improving ME concentrations in each recycling test. Total concentrations of ME with added ectoine were about 1.5 times that without ectoine during five recycling tests (molar ratio of cottonseed oil to methanol, 1:4). Enzymatic reaction kinetics showed, in the concentration ranges of 0.8–1.14 mol/l and 0.03–8 mol/l for triglyceride and methanol, respectively, that ectoine had no effect on the initial reaction rates when methanol concentrations were below 0.5 mol/l. When methanol concentration exceeded 0.5 mol/l, the addition of 0.8 mmol/l ectoine increased the initial reaction rates, and the lipase exhibited a lower affinity for methanol and higher affinity for triglyceride (kinetic parameters of K_mA increase, K_mTG decrease). However, the initial reaction rates decreased significantly when 8 mmol/l ectoine was added, with the lipase having higher affinity for methanol and lower affinity for triglyceride (K_mA decrease, K_mTG increase). The supplementation of ectoine provided a new method for the purpose of improving yield of biodiesel catalyzed by enzyme.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Biodiesel production from sunflower,soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer

This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel) from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM) as a low-cost biocatalyst. The research is focused on three aspects of the process: (a) MPPM synthesis (monolithic, bead, and powder forms), (b) microporous polymeric biocatalyst (MPPB) preparation by immobilization of lipase onto MPPM, and (c) biodiesel production by MPPB. Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE) based on Taguchi methodology.Microporous polymeric matrix (MPPM) containing aldehyde functional group was synthesized by polyHIPE technique using styrene, divinylbenzene, and polyglutaraldehyde. Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%, 85%, and 89% immobilization efficiencies using bead, powder, and monolithic forms, respectively. Immobilized enzymes were successfully used for the production of biodiesel using sunflower, soybean, and waste cooking oils. It was shown that immobilized enzymes retain their activities during 10 repeated batch reactions at 25 °C, each lasting 24 h. Since the developed novel method is simple yet effective, it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases.     

A flow-through enzymatic microreactor immobilizing lipase based on layer-by-layer method for biosynthetic process: Catalyzing the transesterification of soybean oil for fatty acid methyl ester production

A simple and convenient method was proposed in this paper to develop a flow-through enzymatic micro-reactor made from polytetrafluoroethylene (PTFE). It consisted of the polydopamine layer (functioned as a primer) and layer by layer (LBL) coatings composed of polyethylenimine (PEI) and lipase. The multiple deposition of PEI and lipase was the key factor of increasing the enzyme loading on microreactor. After 8 PEI/lipase layers, enzyme loading on the inner surface of 5-m microchannel reached a maximum (350 μg to 400 μg), compared with approximately 20 μg in single layer. Microreactor with higher enzyme loading was successfully applied on transesterification of soybean oil for effective fatty acid methyl ester (FAME, biodiesel) production. A 95.2% conversion rate of biodiesel can be achieved in 53 min under optimized conditions, instead of a couple of hours in the traditional batch reaction.     

Gene cloning,overexpression and characterization of a novel organic solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05

Although the lipase of Geotrichum candidum has been extensively reported, little attention has been focused on molecular genetic and biochemical characterizations of Galactomyces geotrichum lipases. A lipase gene from G. geotrichum Y05 was cloned from both genomic DNA and cDNA sources. Nucleotide sequencing revealed that the ggl gene has an ORF of 1692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The amino acid sequence of this lipase showed 86% identity to lipase of Trichosporon fermentans WU-C12. The mature lipase gene was subcloned into pPIC9K vector, and overexpressed in methylotrophic Pichia pastoris GS115. Active lipase was accumulated to the level of 100.0 U/ml (0.4 mg/ml) in the shake-flask culture, 10.4-fold higher than the activity of the original strain (9.6 U/ml). This yield dramatically exceeds that previously reported with 23–50 U/ml, 0.06 mg/ml and 0.2 mg/ml. The purified lipase exhibited several properties of significant industrial importance, such as pH and temperature stability, wide organic solvent tolerance and broad hydrolysis on vegetable oils. Such a combination of properties makes it a promising candidate for its application in non-aqueous biocatalysis, such as biodiesel production, selective hydrolysis or esterification for enrichment of PUFAs and oil-contaminated biodegradation, which have been drawn considerable attention currently.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.