Production of fructooligosaccharides by mycelium-bound transfructosylation activity present in Cladosporium cladosporioides and Penicilium sizovae

Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fructooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF₂15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C. cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC–PAD chromatography. C. cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), ¹F-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NMR analysis was identified as blastose [Fru-β(2 → 6)-Glc]. P. sizovae was very selective for the formation of ¹F-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.     

Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on ---NH₂ groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m², respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes.     

Improvement of chloroperoxidase stability by covalent immobilization on chitosan membranes

Chloroperoxidase (CPO) from Caldariomyces fumago was optimally covalently immobilized on chitosan membranes pretreated with 0.8 M glutaraldehyde at pH 3.5 to give 3.18 mg CPO g⁻¹ support. Using monochlorodimedone (MCD) as assay substrate, the immobilized-CPO retained 40% activity at 50°C after 40 min whereas free CPO retained only 0.02%. The residual activity for immobilized-CPO was 99 and 58% compared with 68 and 43% for free CPO in the presence of 1.5 M urea and 300 μM H₂O₂, respectively, after 20 h.     

Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005.

The levansucrase gene (lsxA) was cloned from the genomic DNA of Acetobacter xylinum NCI 1005, and the nucleotide sequence of the lsxA gene (1,293 bp) was determined. The deduced amino acid sequence of the lsxA gene showed 57.4% and 46.2% identity with the levansucrases from Zymomonas mobilis and Erwinia amylovora, respectively, while only 35.2% identity with that from Acetobacter diazotrophicus. The gene product of lsxA (LsxA) that was overproduced in E. coli coded for a polypeptide of molecular mass 47 kDa. The LsxA released glucose and produced polysaccharide from sucrose, the structure of which was analyzed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2,6)-linked polyfructan.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Arthrobacter sp. lipase immobilization for improvement in stability and enantioselectivity

Arthrobacter sp. lipase (ABL, MTCC no. 5125) is being recognized as an efficient enzyme for the resolution of drugs and their intermediates. The immobilization of ABL on various matrices for its enantioselectivity, stability, and reusability has been studied. Immobilization by covalent bonding on sepharose and silica afforded a maximum of 380 and 40 IU/g activity, respectively, whereas sol–gel entrapment provided a maximum of 150 IU/g activity in dry powder. The immobilized enzyme displayed excellent stability in the pH range of 4–10 and even at higher temperature, i.e., 50–60°C, compared to free enzyme, which is unstable under extreme conditions. The resolution of racemic auxiliaries like 1-phenyl ethanol and an intermediate of antidepressant drug fluoxetine, i.e., ethyl 3-hydroxy-3-phenylpropanoate alkyl acylates, provided exclusively R-(+) products (∼99% ee, E=646 and 473), compared to cell free extract/whole cells which gave a product with ∼96% ee (E=106 and 150). The repeated use (ten times) of covalently immobilized and entrapped ABL resulted in no loss in activity, thus demonstrating its prospects for commercial applications.     

Improved cosmetic activity by optimizing the Lithospermum erythrorhizon extraction process

This study was conducted to expand the use of Lithospermum erythrorhizon, which is a good source of natural dye, in skin whitening and immune activation cosmetics. The goal was to provide cosmeceutical data about the extraction yield and shikonin contents of this plant by optimizing the ultrasonic extraction and high pressure extraction conditions. Under optimal extraction conditions, which consisted of 500 MPa for 60 min and 120 kHz for 90 min, 27.49 and 3.19 % (w/w) of the highest extraction yield and shikonin contents were obtained, compared to 16.32 and 1.81 % from a conventional ethanol extract (EE) control. Hyaluronidase inhibition activity was measured as 44.24 % after adding 1.0 mg/ml of ethanol extract, but it was as high as 64.19 % when using extract produced by ultrasonication with high pressure extraction (UE + HPE). The MMP-1 expression levels from skin fibroblast cells (CCD-986sk) treated with or without UV irradiation were also lowered by as much as 110.6 % after adding 1.0 mg/ml of the UE + HPE extract, relative to 126.9 % from the EE. After UVA exposure, prostaglandin E2 production from RAW 264.7 was also lower, at 110.6 %, which also indicates that the extract from the UE + HPE process enhanced skin immune activation activities. For the skin whitening activity, tyrosinase inhibitory activity was observed at 67.15 % in the HPE + UE extract, which was ca. 20 % higher than that of the EE extract (57.48 %). To reduce melanin production in Clone M-3 cells, 79.5 % of the melanin production was estimated after adding 1.0 mg/ml of the UE + HPE extract compared to that of the control (no treatment), which was similar to the 77.4 % result found in an ascorbic acid positive control. The highest shikonin secretion was conclusively obtained under the optimal conditions and resulted in a significant improvement of the cosmetic activities of L. erythrorhizon extracts.     

Modifying alcalase activity and stability by immobilization onto chitosan aiming at the production of bioactive peptides by hydrolysis of tilapia skin gelatin

The protease from Bacillus licheniformis, commercially known as Alcalase®, was insolubilized and stabilized by immobilization onto activated chitosan. Activation with different agents, such as glutaraldehyde (GLU-Chi), glyoxyl (GLY-Chi) and divinyl sulfone (DVS-Chi) was investigated. The effect of the immobilization protocol, for instance different pH and times, were also evaluated. GLU-Chi showed the highest activity (35.6U_NPA/g) with the smallest substrate (N-Boc-l-alanine p-nitrophenyl-ester, NPA), while GLY-Chi showed the highest activity (1.5 U_Azocasein/g) using the greatest substrate (azocasein). A 24-h immobilization period was enough to stabilize the enzyme using the three supports under almost all conditions. Operational stability in azocasein hydrolysis was assayed and GLU-Chi showed no activity loss during 5 cycles. DVS-Chi retained around 70 % of its initial activity after the fifth cycle, whereas GLY-Chi activity retained only 10 %. Finally, the biocatalysts were used in the hydrolysis of tilapia skin gelatin aiming the production of peptides with antioxidant activity. The protein hydrolysates obtained using GLU-Chi presented the highest antioxidant activity (36.7 μM Trolox Eq). However, the best results of operational stability were obtained using DVS-Chi, which did not lose its initial activity after 3 consecutive cycles of gelatin hydrolysis.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids

Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp]. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1093–1099, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Enhancement of the activity and enantioselectivity of lipase in organic systems by immobilization onto low-cost support

Lipase from Arthrobacter sp. was immobilized onto low-cost diatomite materials using different protocols for the resolution of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one (HMPC) by asymmetric acylation. The support surface was grafted various functional groups including methacryloxypropyl, vinyl, octyl, dodecyl and γ-(aminopropyl)-glutaraldehyde. These modifications resulted in various mechanisms during the immobilization and thus introduced different characteristics to the prepared lipases. The interfacially adsorbed lipase onto dodecyl-modified support exhibited both higher activity and stability among these immobilized preparations. The modified enzyme-aggregate coating method was performed based on interfacial adsorption in our work, and the characteristics of this immobilized lipase were investigated and compared with those by cross-linking and interfacial adsorption methods. It was shown that the enzyme-aggregate coated lipase yielded the highest activity with a recovered activity of 8.5-fold of the free enzyme, and the highest operational stability with 85% of initial activity remained after 10 recycles. Excellent enantioselectivity (E ≥ 400, with e.e. = 99% of S-HMPC) was obtained for most lipase preparations in our paper (E = 85 for the free enzyme).     

Enhancement of activity of cross-linked enzyme aggregates by a sugar-assisted precipitation strategy: technical development and molecular mechanism

The precipitation of enzyme causes the major activity loss in the conventional protocol for CLEAs preparation. Herein, a sugar-assisted strategy was developed to minimize the activity loss in the step of enzyme precipitation by adding sugar as the stabilizer, which contributed to improve the activity yield of resulting CLEAs. Penicillin G acylase (PGA) was employed as a model enzyme. The effects of glucose, sucrose and trehalose on the activity yields of CLEAs were investigated. The highest activity was obtained in the case of adding trehalose. Confocal laser scanning microscopy and Fourier transform infrared spectroscopy showed that the polar microenvironment and the secondary structure of native enzyme were preserved to some extent when PGA was prepared as sugar-assisted CLEAs, resulting in PGA's higher activity than sugar-free CLEAs. Scanning electron microscope revealed the different inner morphologies, and the kinetic studies showed the higher affinity and resist-inhibition capacity of sugar-assisted CLEAs. Furthermore, stability experiments demonstrated that CLEAs prepared in sugar-assisted strategy remained higher thermal stability when it was incubated at high temperature.     

Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes

Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca2+ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and Futterer, K. (2003) Nat. Struct. Biol. 10, 935-941] has provided evidence for the presence of a bound metal ion, most likely Ca2+. Characterization of site-directed mutants in the putative Ca2+ ion-binding sites of Lb. reuteri Lev and Inu revealed that the Inu Asp520 and Lev Asp500 residues play an important role in Ca2+ binding. Sequence alignments of family GH68 proteins showed that this Ca2+ ion-binding site is (largely) present only in proteins of Gram-positive origin.     

Enhancing the thermal stability of lipases through mutagenesis and immobilization on zeolites

The hydrolysis reaction of p-nitrophenyl butyrate catalyzed by lipases was followed with in situ UV/vis diode array spectrophotometry. Five enzymes - Candida antarctica lipase B and Fusarium solani pisi cutinase wild-type and three single-mutation variants - were tested as catalysts in homogeneous conditions and immobilized on zeolite NaY, on a polyacrylate support and as cross-linked aggregates. Using deconvolution techniques and kinetic modeling, the thermal stability of the different biocatalysts was compared in operational conditions and the results were supported by steady-state enzyme fluorescence measurements. We concluded that both the mutagenesis and the immobilization on zeolite NaY had a positive effect on the thermal stability of F. solani pisi cutinase.     

Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions

In this study raw starch digesting amylase (RSDA) from Aspergillus carbonarius (Bainier) Thom IMI 366159 was stabilized by covalent binding on polyglutaraldehyde (PG), glutaraldehyde (G) activated chitosan beads or post immobilization cross linking of enzyme adsorbed on chitosan. Presence of Ca²⁺ ions (0.5–1.5 mM) activated the PG and G derivatives but repressed the crosslinked enzyme. Optimum pH for cross linked derivative increased by 2 units but was unaltered for PG and G derivatives. Immobilized amylase exhibited improved thermal and storage stability. Immobilized derivatives had no loss of activity after 1 month storage and retained above 90% activity after 10 batch reactions of 60 min each. Immobilization successfully stabilized RSDA and immobilized enzyme from A. carbonarius can be applied in numerous industries for cheap, cost effective and environmentally friendly starch hydrolytic processes to simple sugars.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Selective immobilization of Bacillus subtilis lipase A from cell culture supernatant: Improving catalytic performance and thermal resistance

Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.     

Use of conventional or non-conventional treatments of biochar for lipase immobilization

The objective of this work was to evaluate the influence of dichloromethane (CH₂Cl₂), potassium hydroxide (KOH) and phosphoric acid (H₃PO₄) in the treatment of biochar from guava seeds on conventional and non-conventional techniques (ultrasound and microwave) for the immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption. The effects of the different treatments on the physical and chemical properties of the biochar were evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The immobilization of the BCL on the biochar was evaluated in hydrolysis reactions of olive oil. The conventional treatment of the biochar with KOH showed increased modification on the surface of the biochar, which presented a highly porous surface, and a greater activity for the immobilized biocatalyst compared with the other treatments. The results revealed the potential of biochar as a support novelty for the immobilization of enzymes and for their application in biocatalysis.     

A single mutation within a Ca binding loop increases proteolytic activity,thermal stability,and surfactant stability

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca^2 + ion. This residue lies 15–20 Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate–enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca^2 + and affects the packing of the Ca^2 + binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.     

Peroxidase biocatalysis in water-soluble ionic liquids: activity,kinetic and thermal stability

Abstract

The activity and stability of commercial peroxidase was investigated in the presence of five 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) with either bromide or chloride anions: [C_xmim][X]. The peroxidase activity and stability were better for the shorter alkyl chain lengths of the ILs and peroxidase was more stable in the presence of the bromide anion, rather than chloride. The thermal inactivation profile was studied from 45 to 60 °C in [C₄mim][Cl] and [C₄mim][Br]. The activation energy was also determined. Kinetic analysis of the enzyme in the presence of the [C₄mim][Br] or control (buffer solution) showed that the KM value increased 5-fold and Vm decreased 13-fold in the presence of the IL. The increase in KM indicates that this IL can reduce the binding affinity between substrate and enzyme.