Use Of Biocatalysts Research Articles

Delineation of the complete reaction cycle of a natural Diels-Alderase.

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4 + 2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

Kinetic Characterization of a Lytic Polysaccharide Monooxygenase Reveals a Unique Specificity for Depolymerization at β-O-4 of Lignin Compounds

The intrinsic role of lytic polysaccharide monooxygenases (LPMOs) for oxidative cleavage of a wide range of polysaccharides like cellulose, starch, chitin, and xyloglucan has been studied extensively. Lignin and its compounds are known to act as electron donors for activation of LPMOs. However, the role of LPMOs in the cleavage of lignin compounds and their complete kinetic analysis has not been well understood. Here, an in-depth kinetic characterization was conducted to study the depolymerization of the phenolic β-O-4 lignin compound, guaicyl glycerol β-guaicyl ether (GGE), by LPMO. Ultrahigh-pressure liquid chromatography, thin-layer chromatography, and NMR analysis of the oxidized reaction mixture confirmed the formation of guaiacol from selective cleavage of GGE at β-O-4 linkage by LPMO. Michaelis–Menten and Lineweaver Burk plot kinetics of LPMOs revealed a Km of 3.566 ± 1.002 mM and a catalytic efficiency (kcat/Km) of 43 × 103 M–1 s–1 for GGE. A complete catalytic mechanism involving O2 and H2O2 was proposed and validated for the LPMO-driven cleavage of GGE. The oxidation reactions involving GGE and LPMO in the N2 atmosphere and competitive inhibition studies using catalase confirmed the role of O2 and H2O2 in completing the catalytic cycle of LPMO. Molecular docking and molecular dynamics simulation studies revealed the interaction of amino residues around the copper active site of LPMO with GGE. Per-residue interaction energy of amino acid depicted the maximum interactive energy contribution by MET1 of LPMO. The presence of GGE around the substrate-binding site further confirms the strong interaction of LPMO and GGE. Hence, the interaction between GGE and LPMO confirmed the role of LPMO in cleaving the β-O-4 linkage of lignin, thus making the LPMO an attractive biocatalyst for use in enzymatic cocktails for lignin valorization.

Identification of lipolytic enzymes using high-throughput single-cell screening and sorting of a metagenomic library

The demand for novel, robust microbial biocatalysts for use in industrial and pharmaceutical applications continues to increase rapidly. As a result, there is a need to develop advanced tools and technologies to exploit the vast metabolic potential of unculturable microorganisms found in various environments. Single-cell and functional metagenomics studies can explore the enzymatic potential of entire microbial communities in a given environment without the need to culture the microorganisms. This approach has contributed substantially to the discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples, constructing expression libraries comprising the entire microbial genome, and screening of the libraries for the presence of desired phenotypes. In this study, lipolytic enzymes from the Red Sea were targeted. A high-throughput single-cell microfluidic platform combined with a laser-based fluorescent screening bioassay was employed to discover new genes encoding lipolytic enzymes. Analysis of the metagenomic library led to the identification of three microbial genes encoding lipases based on their functional similarity and sequence homology to known lipases. The results demonstrated that microfluidics is a robust technology that can be used for screening in functional metagenomics. The results also indicate that the Red Sea is a promising, under-investigated source of new genes and gene products.

Eukaryotic Aggresomes: Protocols and Tips for Their Production, Purification , and Handling.

Aggresomes are insoluble protein aggregates found in eukaryotic cells when the intracellular machinery is overtitered by, for example, the overexpression of a recombinant protein. These protein nanoparticles have become excellent models in studies devoted to elucidate protein aggregation processes in eukaryotic cells, like those involved in "conformational disorders" linked to neurodegenerative diseases. Since the presence of such protein aggregates is a hallmark of these conditions, they constitute an excellent target for new therapeutic approaches for such devastating pathologies. Moreover, and following the pathway opened a few years ago by bacterial inclusion bodies, eukaryotic aggresomes have been proposed as a new type of carrier-free, self-immobilized biocatalysts for use in biotechnology and biomedicine. Altogether, unraveling the characteristics and putative applications of naturally occurring protein aggregates has received an increasing interest during the last years. For that, availability of protocols allowing the production and purification of aggresomes constitute a valuable tool to boost research in the abovementioned fields. In this chapter, we describe both upstream and downstream protocols to obtain aggresomes produced in human cells, using as a model the recombinant human enzyme alpha-galactosidase A (GLA), together with technical tips and advices when working and analyzing eukaryotic aggresomes.

Polymerization strategies to produce new polymer biocatalysts for the biodiesel industry

AbstractOne of the major challenges regarding the enzymatic production of biodiesel is the development of more robust, active, and stable immobilized biocatalysts. Thus, the present work aims to develop new enzymatic biocatalysts for use in esterification and transesterification reactions. New polymer supports based on poly(styrene‐co‐divinylbenzene) and poly(methyl methacrylate‐co‐divinylbenzene) platforms were synthesized, incorporating distinct functional compounds into the polymer chains (1‐octene, cardanol, and vinyl benzoate). Two distinct polymerization strategies were adopted for support syntheses: (i) Combined suspension and emulsion polymerization process (core‐shell particles); and (ii) Two‐step polymerization reaction comprising a suspension polymerization in presence of porogenic agents, followed by vacuum application (porous and nonporous particles). The obtained particles were employed for immobilization of lipase B from Candida antarctica. The addition of functional compounds resulted in particles with distinct textural properties. Moreover, particles with 91 m2.g−1 (P(MMA‐co‐DVB)/P(MMA‐co‐DVB)) were produced through combined suspension and emulsion polymerization, whilst particles with 154 m2.g−1 (P[MMA‐co‐DVB‐co‐VB]) were produced through suspension polymerization performed in presence of n‐heptane. Moreover, highly active biocatalysts were produced, leading to esterification conversions above 80%. Thus, based on the performance in esterification and transesterification reactions, the new functional matrices resulted in highly active biocatalysts with good potential for use in biodiesel industry.

The identification and use of robust transaminases from a domestic drain metagenome.

Transaminases remain one of the most promising biocatalysts for use in chiral amine synthesis, however their industrial implementation has been hampered by their general instability towards, for example, high amine donor concentrations and organic solvent content. Herein we describe the identification, cloning and screening of 29 novel transaminases from a household drain metagenome. The most promising enzymes were fully characterised and the effects of pH, temperature, amine donor concentration and co-solvent determined. Several enzymes demonstrated good substrate tolerance as well as an unprecedented robustness for a wild-type transaminase. One enzyme in particular readily accepted IPA as an amine donor giving the same conversion with 2-50 equivalents, as well as being tolerant to a number of co-solvents, and operational in up to 50% DMSO - a characteristic as yet unobserved in a wild-type transaminase. This work highlights the value of using metagenomics for biocatalyst discovery from niche environments, and here has led to the identification of one of the most robust native transaminases described to date, with respect to IPA and DMSO tolerance.

Chitosan hydrogels: A new and simple matrix for lipase catalysed biosyntheses

Lipase from Mucor miehei was immobilized on chitosan hydrogels without using any activation agent. These low-cost and easy to prepare lipase-containing hydrogels could be considered as novel solid-phase biocatalysts for use in organic media. When they were applied as biocatalysts towards the model esterification reaction of propyl laurate synthesis they showed an optimum operation temperature at 30°C and maintained a high catalytic activity in various non-polar organic solvents. The higher initial esterification rates were obtained when biocatalysis took place in branched hydrocarbons where the initial rates recorded were twice the ones observed for straight hydrocarbons, being 0.22mM/min in isooctane and 0.11mM/min in hexane, respectively. The novel catalyst presented excellent reusability, with only 10% loss of activity after 12 uses, which is even higher than the one obtained by lipase-containing chitosan beads prepared with a cross-linker. Moreover, enzyme superactivity was evident for several runs.

Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus.

The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.

Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries.

BackgroundThe use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.ResultsHere we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.ConclusionsThe biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0057-x) contains supplementary material, which is available to authorized users.

Phosphoryl transfer from α-d-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types.

The Cori ester α-d-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s(-1)) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys(189) and Cys(195). Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 (18)O solvent proceeded with complete (18)O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P.

Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including β-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

Extremophilic SHMTs: From Structure to Biotechnology

Recent advances in molecular and structural biology have improved the availability of virtually any biocatalyst in large quantity and have also provided an insight into the detailed structure-function relationships of many of them. These results allowed the rational exploitation of biocatalysts for use in organic synthesis. In this context, extremophilic enzymes are extensively studied for their potential interest for many biotechnological and industrial applications, as they offer increased rates of reactions, higher substrate solubility, and/or longer enzyme half-lives at the conditions of industrial processes. Serine hydroxymethyltransferase (SHMT), for its ubiquitous nature, represents a suitable model for analyzing enzyme adaptation to extreme environments. In fact, many SHMT sequences from Eukarya, Eubacteria and Archaea are available in data banks as well as several crystal structures. In addition, SHMT is structurally conserved because of its critical metabolic role; consequently, very few structural changes have occurred during evolution. Our research group analyzed the molecular basis of SHMT adaptation to high and low temperatures, using experimental and comparative in silico approaches. These structural and functional studies of SHMTs purified from extremophilic organisms can help to understand the peculiarities of the enzyme activity at extreme temperatures, indicating possible strategies for rational enzyme engineering.

Dry Red Wine Making Using Yeast Immobilized on Cork Pieces

The commercial Saccharomyces cerevisiae Uvaferme 299 wine yeast was immobilized on cork pieces to produce a biocatalyst for use in dry red wine making. The immobilized biocatalyst was suitable for clarified and non-clarified grape must fermentation at ambient and low temperatures (0-25 degrees C). Fermentation times were low and very low amounts of residual sugar were detected, showing suitability for dry wine production. The presence of suspended solids in the non-clarified must did not affect the activity of the immobilized cells. Complete fermentation of sugars at 0 degrees C was possible with immobilized Uvaferme 299, although not a cryotolerant strain, indicating a cryoprotective effect of cork. The presence of cork did not affect the composition of major volatiles with methanol and acetaldehyde kept at low levels. Reduction of amyl alcohols on total volatiles was also observed in wines fermented at low temperatures. Differences in the headspace aroma profile in wines produced by immobilized and free cells were found by GC-MS analysis, but no cork taint compounds were detected.

Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes.

BackgroundTermite lignocellulose digestion is achieved through a collaboration of host plus prokaryotic and eukaryotic symbionts. In the present work, we took a combined host and symbiont metatranscriptomic approach for investigating the digestive contributions of host and symbiont in the lower termite Reticulitermes flavipes. Our approach consisted of parallel high-throughput sequencing from (i) a host gut cDNA library and (ii) a hindgut symbiont cDNA library. Subsequently, we undertook functional analyses of newly identified phenoloxidases with potential importance as pretreatment enzymes in industrial lignocellulose processing.ResultsOver 10,000 expressed sequence tags (ESTs) were sequenced from the 2 libraries that aligned into 6,555 putative transcripts, including 171 putative lignocellulase genes. Sequence analyses provided insights in two areas. First, a non-overlapping complement of host and symbiont (prokaryotic plus protist) glycohydrolase gene families known to participate in cellulose, hemicellulose, alpha carbohydrate, and chitin degradation were identified. Of these, cellulases are contributed by host plus symbiont genomes, whereas hemicellulases are contributed exclusively by symbiont genomes. Second, a diverse complement of previously unknown genes that encode proteins with homology to lignase, antioxidant, and detoxification enzymes were identified exclusively from the host library (laccase, catalase, peroxidase, superoxide dismutase, carboxylesterase, cytochrome P450). Subsequently, functional analyses of phenoloxidase activity provided results that were strongly consistent with patterns of laccase gene expression. In particular, phenoloxidase activity and laccase gene expression are mostly restricted to symbiont-free foregut plus salivary gland tissues, and phenoloxidase activity is inducible by lignin feeding.ConclusionTo our knowledge, this is the first time that a dual host-symbiont transcriptome sequencing effort has been conducted in a single termite species. This sequence database represents an important new genomic resource for use in further studies of collaborative host-symbiont termite digestion, as well as development of coevolved host and symbiont-derived biocatalysts for use in industrial biomass-to-bioethanol applications. Additionally, this study demonstrates that: (i) phenoloxidase activities are prominent in the R. flavipes gut and are not symbiont derived, (ii) expands the known number of host and symbiont glycosyl hydrolase families in Reticulitermes, and (iii) supports previous models of lignin degradation and host-symbiont collaboration in cellulose/hemicellulose digestion in the termite gut. All sequences in this paper are available publicly with the accession numbers FL634956-FL640828 (Termite Gut library) and FL641015-FL645753 (Symbiont library).

Construction and expression of an ethanol production operon in Gram-positive bacteria

Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.

Stability of protein-coated microcrystals in organic solvents

Previously we reported a new high activity biocatalyst for use in organic media, termed protein-coated microcrystals (PCMC) [M. Kreiner, B.D. Moore, M.C. Parker, Chem. Commun. 12 (2001) 1906]. These novel biocomposites consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s). Here we have looked at the stability of PCMC and their catalytic behaviour as a function of temperature in different organic media. PCMC show very good long-term stability at room temperature, when stored as suspensions in 1-propanol/1 wt.% H 2O. Candida antarctica lipase B and subtilisin Carlsberg (SC) in PCMC form retained nearly 90% of their initial activity after 1 year at room temperature (RT). The effects of temperature on the catalytic activity of SC-PCMC are solvent-dependant. In 1-propanol/1 wt.% H 2O, the initial rate increased when the temperature was elevated from 25 to 60 °C, whereas in acetonitrile/1 wt.% H 2O, SC-PCMC lost activity. The operational stability of PCMC is also solvent-dependant. In 1-propanol/1 wt.% H 2O, SC-PCMC lost only 16% of the initial activity after five batch cycles. Rather poor stability was found for SC-PCMC in THF/1% (v/v) H 2O and acetonitrile/1% (v/v) H 2O, with a rapid loss of activity within 4 h in a continuous flow reactor. However, during the next 4 days only a slow further deactivation was observed.

Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms.

Modern biotechnology has a steadily increasing demand for vitamins, antibiotics and, in particular, novel biocatalysts for use in the production of flavors, agrochemicals, pharmaceuticals and high-value fine chemicals. Novel experimental approaches are being developed in attempts to identify such molecules. However, it is known that up to 99.8% of the microbes present in many environments are not readily culturable; hence, they cannot be exploited for biotechnology. The 'metagenome technology' offers a solution to this problem by developing culture-independent methods to isolate, clone and express environmental DNA. So far, metagenome-based approaches have led to the isolation of many novel biocatalysts and a variety of other molecules with a high potential for downstream applications.

High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals.

Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.

Immobilization of yeast on dried raisin berries for use in dry white wine-making

Cells of a commercial Saccharomyces cerevisiae strain ( Uvaferme 299) were immobilized on dried raisin berries ( Sultanina variety) to produce an immobilized biocatalyst for use in dry white wine-making. The immobilised biocatalyst was found to be suitable for wine making at ambient temperatures (15–25 °C). The wines produced had low volatile acidities and low methanol and acetaldehyde contents, while volatile by-products showed no statistically significant differences from wines produced by free cells. The immobilized cell system had a good operational stability for more than 4 months. Sensory evaluation revealed differences between wines produced by immobilized and free cells.