Production of prebiotic enriched maple syrup through enzymatic conversion of sucrose into fructo-oligosaccharides.

Maple syrup, a popular natural sweetener has a high content of sucrose, whose consumption is linked to different health issues such as obesity and diabetes. Hence, within this paper, the conversion of sucrose to prebiotics (fructo-oligosaccharides, FOS) was proposed as a promising approach to obtaining a healthier, value-added product. Enzymatic conversion was optimized with respect to key experimental factors, and thereafter derived immobilized preparation of fructosyltransferase (FTase) from Pectinex® Ultra SP-L (FTase-epoxy Purolite, 255 IU/g support) was successfully utilized to produce novel functional product in ten consecutive reaction cycles. The product, obtained under optimal conditions (60 °C, 7.65 IU/mL, 12 h), resulted in 56.0% FOS, 16.7% sucrose, and 27.3% monosaccharides of total carbohydrates, leading to a 1.6-fold reduction in caloric content. The obtained products` prebiotic potential toward the probiotic strain Lactobacillus plantarum 299v was demonstrated. The changes in physico-chemical and sensorial characteristics were esteemed as negligible.

Prebiotic effect of galacto-oligosaccharides on the skin microbiota and determination of their diffusion properties.

OBJECTIVE: Recently, prebiotics are attracting plenty of attention in the field of skin care, since it is found that they are able to support the balance of beneficial and harmful microorganisms on the skin, and accordingly prevent several skin conditions associated with microbial imbalance. Topical application of prebiotics, although insufficiently investigated, holds great promise in improving skin health. The purpose of this research was to determine the prebiotic potential of galacto-oligosaccharides (GOS) for skin microbiota and suitability for incorporation in different topical formulations, and finally, provide insights into their diffusion properties. METHODS: The prebiotic effect of GOS was evaluated through the influence on the growth of Staphylococcus epidermidis and Staphylococcus aureus, the most common resident and pathogenic bacterium of the skin microbiota, respectively. Also, with the future use of GOS in cosmetic products in mind, the diffusion of GOS molecules from two different topical formulations, hydrogel and oil-in-water (O/W) gel emulsion, was monitored employing Franz diffusion cell and two systems-with cellulose acetate membrane and transdermal diffusion test model, Strat-M® membrane. Course of fermentation and the amount of diffused GOS molecules were monitored using high-performance liquid chromatography (HPLC). RESULTS: The in vitro results revealed that GOS at a concentration of 5% (w/v) has a pronounced stimulatory effect on S. epidermidis, while simultaneously showing an inhibitory effect on S. aureus, both in nutrient broth and cosmetic formulations. GOS trisaccharide and tetrasaccharide diffusion coefficients from O/W gel emulsion were calculated to be 5.61·10-6  cm2  s-1 and 1.41·10-8  cm2  s-1 , respectively. The diffusion coefficient of GOS trisaccharides from hydrogel was 3.22·10-6  cm2  s-1 , while it was not determined for tetrasaccharides due to low diffused concentration. Transdermal diffusion tests revealed that GOS incorporated in two formulations stays at the surface of the skin even after 24 h. CONCLUSION: When applied in adequate concentration, GOS has the potential to be used as a skin prebiotic. Novel GOS enriched formulations, Aristoflex® AVC-based hydrogel and Heliogel™-based O/W gel emulsion, provided efficient diffusion and delivery of prebiotic GOS molecules to the skin surface.

OBJECTIF: Récemment, les prébiotiques attirent beaucoup d'attention dans le domaine des soins de la peau, car il a été constaté qu'ils sont capables de soutenir l'équilibre des micro-organismes bénéfiques et nocifs sur la peau et, par conséquent, de prévenir plusieurs affections cutanées associées à un déséquilibre microbien. L'application topique de prébiotiques, bien qu'insuffisamment étudiée, est très prometteuse pour améliorer la santé de la peau. Le but de cette recherche était de déterminer le potentiel prébiotique des galacto-oligosaccharides (GOS) pour le microbiote cutané et leur aptitude à être incorporés dans différentes formulations topiques, et enfin, de fournir des informations sur leurs propriétés de diffusion. MÉTHODES: L'effet prébiotique du GOS a été évalué à travers l'influence sur la croissance de Staphylococcus epidermidis et de Staphylococcus aureus, les bactéries résidentes et pathogènes les plus courantes du microbiote cutané, respectivement. De plus, en gardant à l'esprit l'utilisation future du GOS dans les produits cosmétiques, la diffusion des molécules de GOS à partir de deux formulations topiques différentes, l'hydrogel et l'émulsion de gel huile-dans-eau (H/E), a été surveillée à l'aide d'une cellule de diffusion de Franz et de deux systèmes - avec de la cellulose membrane en acétate et modèle de test de diffusion transdermique, membrane Strat-M®. Le cours de la fermentation et la quantité de molécules de GOS diffusées ont été surveillés en utilisant la chromatographie liquide à haute performance (HPLC). RESULTATS: Les résultats in vitro ont révélé que le GOS à une concentration de 5% (p/v) a un effet stimulant prononcé sur S. epidermidis, tout en montrant simultanément un effet inhibiteur sur S. aureus, à la fois dans les bouillons nutritifs et les formulations cosmétiques. Les coefficients de diffusion GOS trisaccharide et tétrasaccharide de l'émulsion de gel H/E ont été calculés comme étant de 5,61·10−6 cm2 s−1 et 1,41·10-8 cm2 s−1 , respectivement. Le coefficient de diffusion des trisaccharides GOS à partir de l'hydrogel était de 3,22·10−6 cm2 s−1 , alors qu'il n'a pas été déterminé pour les tétrasaccharides en raison de la faible concentration diffusée. Des tests de diffusion transdermique ont révélé que le GOS incorporé dans deux formulations reste à la surface de la peau même après 24 h. CONCLUSION: Lorsqu'il est appliqué à une concentration adéquate, le GOS a le potentiel d'être utilisé comme prébiotique cutané. De nouvelles formulations enrichies en GOS, l'hydrogel à base d'Aristoflex® AVC et l'émulsion de gel H/E à base d'Heliogel™, ont permis une diffusion et une délivrance efficaces des molécules GOS prébiotiques à la surface de la peau.

New Advances in Fabrication of Graphene Glyconanomaterials for Application in Therapy and Diagnosis.

Glycoderivatives are an important class of molecules with enormous relevance in numerous biological phenomena; therefore, they have a key role in the learning, understanding, and assessment of different diseases. Nanotechnology, and in particular the design of new nanomaterials, is one of the areas of greatest interest today. In this case, graphene nanomaterials represent very interesting platforms for studying glycosystems, glyconanomaterials that combine the biomolecular recognition and the characteristics of nanoscale objects in the development of early diagnosis systems, and efficient specific therapeutic modalities. In this mini-review, we discuss some results recently described in the literature on the conjugation of graphene materials and carbohydrates through the selective interaction of glycoenzymes in graphene to create new materials with biosensing applications, the development and application of sugar-graphene composites, and finally biosystems combining the properties of graphene with metallic nanoparticles and sugars for the creation of excellent glyconanomaterials as novel systems for the therapy or diagnosis of important diseases such as cancer or diabetes.

Response Surface Methodology As a New Approach for Finding Optimal MALDI Matrix Spraying Parameters for Mass Spectrometry Imaging.

Automated spraying devices have become ubiquitous in laboratories employing matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in part because they permit control of a number of matrix application parameters that can easily be reproduced for intra- and interlaboratory studies. Determining the optimal parameters for MALDI matrix application, such as temperature, flow rate, spraying velocity, number of spraying cycles, and solvent composition for matrix application, is critical for obtaining high-quality MALDI-MSI data. However, there are no established approaches for optimizing these multiple parameters simultaneously. Instead optimization is performed iteratively (i.e., one parameter at a time), which is time-consuming and can lead to overall nonoptimal settings. In this report, we demonstrate the use a novel experimental design and the response surface methodology to optimize five parameters of MALDI matrix application using a robotic sprayer. Thirty-two combinations of MALDI matrix spraying conditions were tested, which allowed us to elucidate relationships between each of the application parameters as determined by MALDI-MS (specifically, using a 15 T Fourier transform ion cyclotron resonance mass spectrometer). As such, we were able to determine the optimal automated spraying parameters that minimized signal delocalization and enabled high MALDI sensitivity. We envision this optimization strategy can be utilized for other matrix application approaches and MALDI-MSI analyses of other molecular classes and tissue types.

Amino-modified kraft lignin microspheres as a support for enzyme immobilization.

In this research, it has been demonstrated that amino-modified microspheres (A-LMS) based on bio-waste derived material, such as kraft lignin, have good prospects in usage as a support for enzyme immobilization, since active biocatalyst systems were prepared by immobilizing ß-galactosidase from A. oryzae and laccase from M. thermophila expressed in A. oryzae (Novozym® 51003) onto A-LMS. Two types of A-LMS were investigated, with different emulsifier concentrations (5 wt% and 10 wt%), and microspheres produced using 5 wt% of emulsifier (A-LMS_5) showed adequate pore shape, size and distribution for enzyme attachment. The type of interactions formed between enzymes (ß-galactosidase and laccase) and A-LMS_5 microspheres demonstrated that ß-galactosidase is predominantly attached via electrostatic interactions while attachment of laccase is equally governed by electrostatic and hydrophobic interactions. Furthermore, the A-LMS_5-ß-galactosidase exhibited specificity towards recognized prebiotics (galacto-oligosaccharides (GOS)) synthesis with 1.5-times higher GOS production than glucose production, while for environmental pollutant lindane degradation, the immobilized laccase preparation exhibited high activity with a minimum remaining lindane concentration of 22.4% after 6 days. Thus, this novel enzyme immobilization support A-LMS_5 has potential for use in green biotechnologies.

Evaluation of ß-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization.

ß-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude ß-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that ß-galactosidase from L. acidophilus shows a particular specificity towards the formation of ß-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 g/l GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50°C).

Immobilization of dextransucrase on functionalized TiO2 supports.

The TiO2 based hybrid supports with different functional groups (amino, glutaraldehyde or epoxy) were prepared and their influence on immobilization of dextransucrase (DS) was studied. Novel synthetic route for surface modification of TiO2 with amino and glutaraldehyde groups was developed taking advantage of charge transfer complex (CTC) formation between surface Ti atoms and salicylate-type of ligand (5­aminosalicylic acid (5-ASA)). The proposed coordination of 5-ASA to the surface of TiO2 powder and optical properties of CTC was presented. The pristine TiO2 and amino functionalized TiO2 have higher sorption capacity for DS (12.6 and 12.0mgg-1, respectively) compared to glutaraldehyde and epoxy activated supports (9.6 and 9.8mgg-1, respectively). However, immobilized enzyme to either glutaraldehyde or epoxy functionalized TiO2 have almost two times higher expressed activities compared to pristine TiO2 support (258, 235 and 142IUg-1, respectively). Thermal stability of enzyme immobilized on glutaraldehyde and epoxy functionalized supports was studied at 40°C, as well as operational stability under long-run working conditions in repeated cycles. After five cycles, DS imobilized on glutaraldehyde activated support retained almost 70% of its initial expressed activity, while, after five cycles, performance of DS immobilized on epoxy activated support was significantly lower (15%).

Covalent Immobilization of Enzymes on Eupergit® Supports: Effect of the Immobilization Protocol.

A selection of best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on an industrial level. Eupergit® supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is advantageous for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads. The modification of the conventionally immobilized enzyme with ethylenediamine via the carbodiimide route seems to be a powerful tool to improve its stability and catalytic activity.

Synthesis of medium-chain length capsinoids from coconut oil catalyzed by Candida rugosa lipases.

A commercial preparation of Candida rugosa lipases (CRL) was tested for the production of capsinoids by esterification of vanillyl alcohol (VA) with free fatty acids (FA) and coconut oil (CO) as acyl donors. Screening of FA chain length indicated that C8-C12 FA (the most common FA found in CO triglycerides) are the best acyl-donors, yielding 80-85% of their specific capsinoids. Hence, when CO, which is rich in these FA, was used as the substrate, a mixture of capsinoids (vanillyl caprylate, vanillyl decanoate and vanillyl laurate) was obtained. The findings presented here suggest that our experimental method can be applied for the enrichment of CO with capsinoids, thus giving it additional health promoting properties.

Immobilization of Candida antarctica lipase B onto Purolite® MN102 and its application in solvent-free and organic media esterification.

The aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite® MN102 and to apply immobilized catalyst for the enzymatic synthesis of two valuable esters-isoamyl acetate and L-ascorbyl oleate. At optimized conditions (1 M phosphate buffer pH = 7, 7 h at 25 °C, and 18.75 mg of offered proteins g-1 of support), immobilized lipase with hydrolytic activity of 888.4 p-nitrophenyl butyrate units g-1 was obtained. Afterwards, preparation was applied for the solvent-free synthesis of isoamyl acetate from triacetin and isoamyl alcohol. At 75 °C, 1 M of isoamyl alcohol, and 6 mg ml-1 of enzyme 100 % yield was achieved in 6 h, while at prolonged reaction times, complete conversion was enabled even at lower temperatures, lower lipase loadings, and higher substrate concentrations. After 15 consecutive reuses (60 h), activity of catalyst dropped to 50 % of its initial value and total amount of 1.31 mol (170.55 g) of ester with 1 g of biocatalyst was produced. Even higher operational stability of lipase (25 % loss of activity in 200 h) was observed in the synthesis of L-ascorbyl oleate performed in organic solvent (t-butanol). Multiple use of one batch of immobilized biocatalyst in both cases led to a significant process cost reduction and substantial increment of corresponding productivities.

Structural Elucidation of Enzymatically Synthesized Galacto-oligosaccharides Using Ion-Mobility Spectrometry-Tandem Mass Spectrometry.

Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with ß-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of ß-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry-tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that ß-galactosidase from Aspergillus oryzae has the highest affinity toward formation of ß-(1→3) or ß-(1→6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis.

Design of biocompatible immobilized Candida rugosa lipase with potential application in food industry.

BACKGROUND: Biocatalysts are a promising alternative for the production of natural flavor compounds. Candida rugosa lipase (CRL) is a particularly important biocatalyst owing to its remarkable efficiency in both hydrolysis and synthesis. However, additional stabilization is necessary for successful industrial implementation. This study presents an easy and time-saving method for immobilizing this valuable enzyme on hydroxyapatite (HAP), a biomaterial with high protein-binding capacity. RESULTS: Targeted immobilized CRL was obtained in high yield of ≥98%. Significant lipase stabilization was observed upon immobilization: at 60 °C, immobilized lipase (HAP-CRL) retained almost unchanged activity after 3 h, while free CRL lost 50% of its initial activity after only 30 min. The same trend was observed with tested organic solvents. Methanol and hexane had the most pronounced effect: after 3 h, only HAP-CRL was stable and active, while CRL was completely inactivated. The practical value of the prepared catalyst was tested in the synthesis of the aroma ester methyl acetate in hexane. Reaction yields were 2.6 and 52.5% for CRL and HAP-CRL respectively. CONCLUSION: This research has successfully combined an industrially prominent biocatalyst, CRL, and a biocompatible, environmentally suitable carrier, HAP, into an immobilized preparation with improved catalytic properties. The obtained CRL preparation has excellent potential for the food and flavor industries, major consumers in the global enzyme market. © 2016 Society of Chemical Industry.

Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity.

Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco-friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase-catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short-chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L(-1) of lipase, 50 mM of vitamin C, 10-fold molar excess of oleic acid, and 0.7 mL L(-1) of initial water. Obtained results showed that even short- and medium-chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l-ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes.

Dual effect of benzyl alcohol on α-glucosidase activity: efficient substrate for high yield transglucosylation and non-competitive inhibitor of its hydrolytic activity.

Benzyl alcohol, a potent anesthetic and bacteriostatic, can be efficiently glucosylated by α-glucosidase from Saccharomyces cerevisiae to produce benzyl alcohol α-glucoside with a 75% yield. However, while studying the transglucosylation reaction conditions, it was found out that benzyl alcohol is a non-competitive inhibitor of α-glucosidase's hydrolytic activity (Ki=18mM, toward maltose). Due to its interesting ability to be glycosylated by the enzyme and to inhibit its hydrolytic activity, we proposed a plausible mechanism for the phenolic α-glucosydase inhibitor's binding, since the mechanism of inhibition has not yet been elucidated.

Lipase-catalyzed synthesis of ascorbyl oleate in acetone: optimization of reaction conditions and lipase reusability.

Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60°C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4(th) hour of reaction enabled increase of yield from 16.7 mmol g⁻¹ to 19.3 mmol g⁻¹. Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation.

Synthesis of aliphatic esters of cinnamic acid as potential lipophilic antioxidants catalyzed by lipase B from Candida antarctica.

Immobilized lipase from Candida antarctica (Novozyme 435) was tested for the synthesis of various phenolic acid esters (ethyl and n-butyl cinnamate, ethyl p-coumarate and n-butyl p-methoxycinnamate). The second-order kinetic model was used to mathematically describe the reaction kinetics and to compare present processes quantitatively. It was found that the model agreed well with the experimental data. Further, the effect of alcohol type on the esterification of cinnamic acid was investigated. The immobilized lipase showed more ability to catalyze the synthesis of butyl cinnamate. Therefore, the process was optimized for the synthesis of butyl cinnamate as a function of solvent polarity (logP) and amount of biocatalyst. The highest ester yield of 60.7 % was obtained for the highest enzyme concentration tested (3 % w/w), but the productivity was for 34 % lower than the corresponding value obtained for the enzyme concentration of 1 % (w/w). The synthesized esters were purified, identified, and screened for antioxidant activities. Both DPPH assay and cyclic voltammetry measurement have shown that cinnamic acid esters have better antioxidant properties than cinnamic acid itself.

Specificity of maltase to maltose in three different directions of reaction: hydrolytic, vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis.

Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping-pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α-isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that maltase has interestingly high K(m) value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate.

Novel glycoside of vanillyl alcohol, 4-hydroxy-3-methoxybenzyl-α-D-glucopyranoside: study of enzymatic synthesis, in vitro digestion and antioxidant activity.

Novel glucoside of physiological active vanillyl alcohol was synthesized for the first time using maltase from Saccharomyces cerevisiae as catalyst, and established its structure as 4-hydroxy-3-methoxybenzyl-α-D: -glucopyranoside. The key reaction factors for this transglucosylation reaction were optimized using response surface methodology and the highest yield so far in maltase catalyzed transglucosylation reaction was obtained. It was found out that optimum temperature of reaction was 37 °C, optimal maltose concentration was 60% (w/v), optimal pH was 6.6, and optimal concentration of vanillyl alcohol was 158 mM. Under these conditions, yield of glucoside was 90 mM with no by product formation. It was shown that this compound posses good antioxidant activity as well as stability in gastrointestinal tract. It was demonstrated that it is hydrolyzed on brush border membrane of enterocytes, so it can serve in protecting gastrointestinal system from oxidation, as well as source of anticonvulsive drug after the hydrolysis of glucoside on brush border membrane of small intestine.

One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite.

Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83 U/mg specific activity. Silver staining after SDS-PAGE revealed the presence a single band of 45 kDa. The enzyme exhibited a temperature optimum of 60°C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+)) and stimulated by 50mM Fe(2+). Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect.

Adding value to the oil cake as a waste from oil processing industry: production of lipase and protease by Candida utilis in solid state fermentation.

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level-three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g(-1) and a protease activity value of 110 U g(-1) were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.