Novel chitosan membranes as support for lipases immobilization: Characterization aspects

Membranes of chitosan (QS), chitosan treated with glutaraldehyde (QGA) and chitosan crown ether (QCE) were utilized as carriers for immobilization of Candida antarctica and Candida rugosa lipases. Membrane supports were characterized by several techniques (Raman spectroscopy, elemental analysis by CHN determination and Energy Dispersive X-ray (EDX), water sorption isotherms, and surface area from nitrogen sorption data). To verify the presence of enzymes, some of these techniques were also used for lipase on chitosan biocatalytic systems. Measurements of protein load from Biuret assays and catalytic activity in esterification in nonaqueous media were also made for the immobilized enzymes. Sorption isotherms at 20, 30, 40 and 50 °C for QS, QGA and QCE supports were fitted to the Guggenheim, Anderson and Böer model. GAB monolayer moisture parameter, Xm, varied between 0.029 and 0.051 for QS, 0.039 and 0.058 for QGA and 0.039–0.075 g of water g⁻¹ s.s. for QCE membranes. Elemental analysis and Raman spectra measurements of the lipase, supports and immobilized lipase systems gave evidence of the presence of enzymes on supports. Chitosan supports with internal surface area (m2 g⁻¹) among 3.31 and 1.26 were obtained. Regardless of these low values, acceptable protein load (0.61 to 3.21%) and esterification initial rates were achieved (0.88–2.75 mmol min⁻¹ g of protein⁻¹).     

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

Effects of oils and oil-related substrates on the synthetic activity of membrane-bound lipase from Rhizopus chinensis and optimization of the lipase fermentation media

The lipase from filamentous fungi Rhizopus chinensis, as a membrane-bound enzyme, possesses the excellent catalysis ability for esterification and transesterification reactions, and has a good potential in many industrial applications. In order to improve the synthetic activity of the lipase, the effects of oils and oil-related substrates on its production and the fermentation media optimization were investigated. Based on the results, it was suggested that oleic acid could be the important substrate for the lipase production. Among various oils and oil-related substrates, olive oil containing high content of oleic acid was the optimal one for the lipase production. Using orthogonal test and response surface methodology (RSM), the composition of fermentation media was further optimized. The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g L⁻¹, olive oil 21.94 and 22.99 g L⁻¹, maltose 12.91 and 14.34 g L⁻¹, respectively, with K₂HPO₄ 3 g L⁻¹, MgSO₄·7H₂O 5 g L⁻¹ and initial pH 6.0. Under the optimal conditions, the lipase activity and the activity yield were improved 61.5 and 93.4% comparing the results before optimization, respectively. The adequate models obtained had predicted the lipase production successfully.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Alternative production process strategies in E. coli improving protein quality and downstream yields

Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g⁻¹ DCW, but the final specific enzyme activity (1.7 AU mg⁻¹ RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield:

(i) Temperature reduction to 28 °C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g⁻¹ DCW. The enzyme activity increased to 4 AU mg⁻¹ RhuA and purification-immobilization yield to 93%.

(ii) A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g⁻¹ DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg⁻¹ RhuA, and 95% of immobilization yield was achieved.

For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi

Effects of carbon concentration and carbon to nitrogen (C:N) ratio on six biocontrol fungal strains are reported in this paper. All fungal strains had extensive growth on the media supplemented with 6–12 g l⁻¹ carbon and C:N ratios from 10:1 to 80:1, and differed in nutrient requirements for sporulation. Except for the two strains of Paecilomyces lilacinus, all selected fungi attained the highest spore yields at a C:N ratio of 160:1 when the carbon concentration was 12 g l⁻¹ for Metarhizium anisopliae SQZ-1-21, 6 g l⁻¹ for M. anisopliae RS-4-1 and Trichoderma viride TV-1, and 8 g l⁻¹ for Lecanicillium lecanii CA-1-G. The optimal conditions for P. lilacinus sporulation were 8 g l⁻¹ carbon with a C:N ratio of 10:1 for M-14 and 12 g l⁻¹ carbon with a C:N ratio of 20:1 for IPC-P, respectively. The results indicated that the influence of carbon concentration and C:N ratio on fungal growth and sporulation is strain dependent; therefore, consideration for the complexity of nutrient requirements is essential for improving yields of fungal biocontrol agents.     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

Enzymatic pre-hydrolysis applied to the anaerobic treatment of effluents from poultry slaughterhouses

A pool of hydrolases with 21.4 U g⁻¹ lipase activity was produced through solid-state fermentation of the fungus Penicillium restrictum in waste from the Orbignya oleifera (babassu) oil processing industry. Enzymatic hydrolysis and anaerobic biodegradability tests were conducted on poultry slaughterhouse effluents with varying oil and grease contents (150–1200 mg l⁻¹) and solid enzymatic pool concentrations (0.1–1.0% w/v). Enhanced anaerobic treatment efficiency relative to raw effluent was achieved when a 0.1% concentration of enzymatic pool was used in the pre-hydrolysis stage with 1200 mg oil and grease l⁻¹ (chemical oxygen demand (COD) removal efficiency of 85% vs. 53% and biogas production of 175 ml vs. 37 ml after 4 d).     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Entropy decrease associated to solute compartmentalization in the cell

We have deduced equations to quantify the entropy associated to the compartmentalization of components in eukaryotic cells as a function of cell and compartment volumes, and of the concentration of solutes. On the basis of known and plausible values of volume and solute concentrations and the deduced equations, we estimate that the contribution of compartmentalization to the decrease of entropy is approximately −14.4 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−0.7 J K⁻¹ L⁻¹) in the case of Saccharomyces cerevisiae, a typical eukaryotic cell, and approximately −49.6 × 10⁻¹⁴ J K⁻¹ cell⁻¹ (−1.0 J K⁻¹ L⁻¹) in the more complex Chlamydomonas reinhardtii. When compared with other potential contributing factors, such as the informational entropy of DNA and the conformational entropy of proteins, compartmentalization appears as an essential development that significantly decreased the entropy of living cells during biological evolution.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O₂ h^− 1 g^− 1; summer: 0.31 ± 0.06 μmol O₂ h^− 1 g^− 1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH⁻). The A / AH⁻ ratio showed minimum values in winter (3.7 ± 0.2 10^− 5 AU) and increased in summer (18 ± 5 10^− 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg^− 1 fresh mass [FW] in winter and 314 ± 45 pmol mg^− 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg^− 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg^− 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Studies on a novel carbon source and cosolvent for lipase production by<Emphasis Type="Italic"> Candida rugosa</Emphasis>

Oleic acid esters were shown to be the best carbon source for both cell growth and lipase production by Candida rugosa. Use of a cosolvent, dodecane, in fermentations improved the solubility of solid substrates and increased oxygen solubility. This resulted in the highest lipase activity in batch fermentation with glycerol trioleate and dodecane. Lipase activity reached 77.1 units ml^–1.