Production Of Industrial Enzymes Research Articles

Ability of industrial dyes decolorization and ligninolytic enzymes production by different Pleurotus species with special attention on Pleurotus calyptratus, strain CCBAS 461

Eight different Pleurotus species were tested for their Orange G and Remazol Brilliant Blue R (RBBR) decolorization capacity and their ligninolytic properties. All the species produced laccase (Lac) and manganese peroxidase (MnP) and in five species we found aryl-alcohol oxidase (AAO) activity. Strain CCBAS 461 of a very little studied species Pleurotus calyptratus was chosen for a more detailed study. This strain produced a relatively high amount of Lac, MnP and also AAO. Within 14 days the strain decolorized up to 91% of Orange G and 85% of RBBR in liquid culture and more than 50% of these dyes on agar plates. P. calyptratus is able to decolorize efficiently also other azo and phthalocyanine dyes, but only a limited decolorization capacity was found in the case of polyaromatic and triphenylmethane dyes. Lac and MnP production was strongly influenced by the kind of cultivation media and by the dye present.

Manganese peroxidase from the white‐rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Inulinase production using garlic (Allium sativum) powder as a potential substrate in Streptomyces sp.

Inulinase production, using various naturally occurring inulin-containing agricultural plants as carbon sources and pure inulin (chicory, Sigma, Chem., Co., USA), was studied with an indigenously isolated actinomycete strain. All substrates depicted some activity but profile varied among them. Maximum inulinase activity was observed with garlic (Allium sativum) (524IU/L). The enzyme activity was about 1.6-fold higher than activity obtained by using pure inulin as a carbon source. This is quite interesting attractive feedstock for large-scale fermentation, whereas pure inulin (chicory, Sigma, Chem., Co., USA) is only available in limited quantities and at very high cost. The present study suggests a significant impact of their use in industrial processes, considering the high cost of commercially pure inulin. The optimum temperature and pH for enzyme activity were 60°C and 6.0, respectively. All metal salts except NaHCO3, AgNO3 and CoCl2 were not well tolerated and did adversely affect inulinase activity. The effect of thermal stabilizers on inulinase activity was studied. Consequently, glycerol and mannitol have been shown to have a protective effect on enzyme activity. All these conditions make Streptomyces sp., a potential candidate for industrial enzymatic production of high fructose syrup and in other large-scale biotechnological processes.

The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface-active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae was cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the rolA gene encoding hydrophobin RolA was highly transcribed. High levels of RolA and its localization on the cell surface in the presence of PBSA were confirmed by immunostaining. Under these conditions, A. oryzae simultaneously produced the cutinase CutL1, which hydrolyses PBSA. Pre-incubation of PBSA with RolA stimulated PBSA degradation by CutL1, suggesting that RolA bound to the PBSA surface was required for the stimulation. Immunostaining revealed that PBSA films coated with RolA specifically adsorbed CutL1. Quartz crystal microbalance analyses further demonstrated that RolA attached to a hydrophobic sensor chip specifically adsorbed CutL1. Circular dichroism spectra of soluble-state RolA and bound RolA suggested that RolA underwent a conformational change after its adsorption to hydrophobic surfaces. These results suggest that RolA adsorbed to the hydrophobic surface of PBSA recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis. A fluorescence recovery after photobleaching experiment on PBSA films coated with FITC-labelled RolA suggested that RolA moves laterally on the film. We discuss the novel molecular functions of RolA with regard to plastic degradation.

Detección de proteasas extracelulares en especies de Mucor

The fungi are characterized by their abilities to produce and secrete enzymes to the external environment. The species of genus Mucor are a group of fungal microbes with important biotechnological potential, which are responsible for production of industrial enzymes. This work evaluated the ability of protease production in twelve species of genus Mucor. The strains were kept for 120 h under the incubation temperature of 28 degrees C on a shaker at 120 rpm. The detection of proteolytic activity was evaluated in all species, the higher activity was detected in Mucor racemosus Fres. f. chibinensis (Neophytova) Schipper.

An Overview of the CCAAT-Box Binding Factor in Filamentous Fungi: Assembly, Nuclear Translocation, and Transcriptional Enhancement

Filamentous fungi are frequently used for the production of industrial enzymes, since they produce a variety of enzymes including polysaccharide-degrading enzymes. Among the many filamentous fungi, Aspergillus species, such as A. oryzae and A. niger, are known as strong producers of amylolytic enzymes. We have been studying on the regulatory mechanisms underlying the expression of A. oryzae amylolytic genes. Based on analyses using a hybrid model system of A. nidulans transformed by a gene encoding A. oryzae Taka-amylase A, the major amylase (taaG2), we have found that three factors, CCAAT-box binding protein, CreA, and AmyR, are involved in taaG2 gene expression and regulation. In this review, the focus is on the CCAAT-box binding protein of filamentous fungi. The assembly, nuclear translocation, and transcriptional enhancement mechanisms of the CCAAT-box binding protein are discussed.

From fundamental studies of sporulation to applied spore research

Sporulation in the Gram-positive bacterium, Bacillus subtilis, has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although many basic aspects of this process are now understood in great detail, including the crystal and NMR structures of some of the key proteins and their complexes, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or are only described by 'controversial' models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely be increased if we were to include sporulation processes in the Gram-negative spore formers. Spore formers have great potential in applied research. They have been used for many years as biodosimeters and as natural insecticides, exploited in the industrial production of enzymes, antibiotics, used as probiotics and, more, exploited as possible vectors for drug delivery, vaccine antigens and other immunomodulating molecules. This report describes these and other aspects of current fundamental and applied spore research that were presented at European Spores Conference held in Smolenice Castle, Slovakia, June 2004.

Improved xylanase production by Trichoderma reesei grown on l-arabinose and lactose or d-glucose mixtures

Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with D-glucose or lactose. The fungal cells consumed all aldopentoses tested, except L-xylose and L-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on L-arabinose as the main carbon source. The total xylanase activity produced by cells grown on L-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of D-glucose (15 g l(-1)) and L-arabinose (5 g l(-1)), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only D-glucose (20 g l(-1)). In a similar experiment, in which cells were grown on a mixture of lactose and L-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of L-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture.

Production of Industrial Enzymes (Amylase, Carboxymethylcellulase and Protease) by Bacteria Isolated from Marine Sedentary Organisms

AbstractThe abilities of bacteria isolated from eight marine sedentary organisms, six marine sponges (Spirastrella sp., Phyllospongia sp., Ircinia sp., Aaptos sp., Azorica sp. and Axinella sp.), one soft coral (Lobophytum sp.) and one alga (Sargassum sp.) to produce industrial enzymes (amylase, carboxymethylcellulase and protease) were examined. The mean total viable counts of the bacterial isolates ranged from 8.7 × 104 to 8.4 × 105 cfu/g wet weight of the organism. All eight organisms harboured amylase (0.05–0.5 IU/ml), carboxymethylcellulase (0.05–0.5 IU/ml) and protease (0.1–0.5 IU/ml) producing bacteria. Of 56 bacterial strains tested, as many as 60 to 83% of the strains produced at least one of the three enzymes, and 47% of strains were able to produce all three enzymes. High activities (> 0.5 IU/ml) of the three enzymes were recorded in bacterial strains belonging to the genera Alcaligenes and Bacillus. From the results of this study, it appears that bacteria associated with marine sedentary organisms are the novel source of industrial enzymes for possible commercial applications and may play an important role in enzyme‐catalysed organic matter cycling in marine environments.

Perspectives for the industrial enzymatic production of glycosides.

Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus, enzyme-catalyzed reactions are a good alternative. However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product. Unfortunately, a bioreactor for the commercial scale production of glycosides is not available. The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.

Effect of media supplements and culture conditions on inulinase production by an actinomycete strain.

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.

From green plants to industrial enzymes

Transgenic plants provide a viable technology for producing industrial proteins. Advantages of plant production systems include low cost of goods, stable protein storage in seeds, ease and speed of scale-up and the possibility of direct addition of plant material to industrial processes. Using transgenic plants for production of proteins includes several steps designed to result in high expression of foreign proteins—from gene manipulation to breeding. Characteristics of four protein examples are presented —β glucuronidase, avidin, laccase and trypsin. These proteins represent a range of molecular weights, activities, and localizations, demonstrating the versatility of the system. The benefits of transgenic plant technology for industrial enzyme production include replacement of chemical processes that cause environmental pollution.

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and α-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and α-amylase (AMY) on five carbon sources that have different reduction degrees ( γ), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose ( γ=4.0), acetate ( γ=4.0), and the TCA cycle organic-acids succinate ( γ=3.5), malate ( γ=3.0), and citrate ( γ=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates ( μ) were calculated as 1.142 and 0.766 h −1, respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191 mmol g −1 DW h −1 when glucose uptake rate was 10 mmol g −1 DW h −1 and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.

Regulation of the amylolytic and (hemi-)cellulolytic genes in aspergilli.

Filamentous fungi produce high levels of polysaccharide-degrading enzymes and are frequently used for the production of industrial enzymes. Because of the high secretory capacity for enzymes, filamentous fungi are effective hosts for the production of foreign proteins. Genetic studies with Aspergillus nidulans have shown pathway-specific regulatory systems that control a set of genes that must be expressed to catabolize particular substrates. Besides the pathway-specific regulation, wide domain regulatory systems exist that affect a great many individual genes in different pathways. A molecular analysis of various regulated systems has confirmed the formal models derived from purely genetic data. In general, many genes are subject to more than one regulatory system. In this article, we describe two transcriptional activators, AmyR and XlnR, and an enhancer, Hap complex, in view of their regulatory roles in the expression of the amylolytic and (hemi-)cellulolytic genes mainly in aspergilli. The amyR gene has been isolated as a transcriptional activator involved in the expression of amylolytic genes from A. oryzae, A. niger, and A. nidulans, and the xlnR gene, which has been isolated from A. niger and A. oryzae, activates the expression of xylanolytic genes as well as some cellulolytic genes in aspergilli. Both AmyR and XlnR have a typical zinc binuclear cluster DNA-binding domain at their N-terminal regions. Hap complex, a CCAAT-binding complex, enhances the overall promoter activity and increases the expression levels of many fungal genes, including the Taka-amylase A gene. Hap complex comprises three subunits, HapB, HapC, and HapE, in A. nidulans and A. oryzae as well as higher eukaryotes, whereas HAP complex in Saccharomyces cerevisiae and Kluyveromyces lactis has the additional subunit, Hap4p, which is responsible for the transcriptional activation. Hap complex is suggested to enhance transcription by remodeling the chromatin structure. The regulation of gene expression in filamentous fungi of industrial interest could follow basically the same general principles as those discovered in A. nidulans. The knowledge of regulation of gene expression in combination with traditional genetic techniques is expected to be increasingly utilized for strain breeding. Furthermore, this knowledge provides a basis for the rational application of transcriptional regulators for biotechnological processes in filamentous fungi.

INFLUENCE OF INOCULUM SIZE ON PHYTASE PRODUCTION AND GROWTH IN SOLID–STATE FERMENTATION BY ASPERGILLUS NIGER

Solidstate fermentation is experiencing renewed interest for industrial enzyme production. Previous studies onthe effects of fungal inoculum size on product yield have focused on spore inoculum. However, some organisms requirevegetative inocula. This study investigated the effects of initial inoculum colony age, vegetative inoculum size, and durationof fermentation on the production of fungal biomass and phytase in solidstate fermentation using Aspergillus niger grownon wheat bran and soy meal. Initial inocula from 7 and 14dayold potato dextrose agar (PDA) plates were used to studythe effect of inoculum colony age in liquid culture and its further influence on fermentation. The liquid inoculum size of (60 480 mg of biomass/5 g substrate) and the duration of fermentation were studied for 10 days with periodic sampling every48 hours. The study was conducted as a replicated full factorial experiment. Statistical analysis showed that phytaseproduction and growth were not affected by inoculum size (over 60 mg/5 g substrate) after 48 hours of fermentation. Theduration of fermentation was highly significant with a maximal phytase (1001
94 U/g substrate) and biomass (0.032
0.007g glucosamine/g substrate) formation at 192 h of SSF. Phytase yield and biomass formation were strongly correlated for the7 and 14day plate cultures (the estimated correlation coefficients being 0.87 and 0.73), indicating that phytase productionis strongly growthassociated.

Production of pectolytic enzymes – a review

Pectolytic enzymes play an important role in food processing industries and alcoholic beverage industries. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. These enzymes are mainly synthesized by plants and microorganisms. Aspergillus niger is used for industrial production of pectolytic enzymes. This fungus produces polygalacturonase, polymethylgalacturonase and pectinlyase. This review mainly concerns with the production of pectolytic enzymes using different carbon sources. It also deals with the effect of operating parameters such as temperature, aeration rate, agitation and type of fermentation on the production of these enzymes.

Media Evaluation for the Production of Microbial Enzymes

This study compares the performances of animal-based byproducts with other complex nutrients. Productions of α-amylase by Bacillus subtilis, glucoamylase by Aspergillus niger, endocellulase, exocellulase, and xylanase by Trichoderma viride, protease by B. subtilis, and B. licheniformis were evaluated in shake-flask fermentation. Culture broths were analyzed daily for enzyme activity for up to 9 days. Significant increases in α-amylase, cellulase, and xylanase activities were observed. Data strongly suggest that many of these byproducts can be used to replace the more expensive complex nutrients in industrial enzyme production with a corresponding increase in enzyme production. Keywords: Enzyme production; α-amylase; glucoamylase; endocellulase; exocellulase; xylanase; protease

Aspects of the use of complex media for submerged fermentation of Aspergillus awamori

For the industrial production of enzymes using fungi, often complex media containing solid substrates are used. In this study, fermentations with Aspergillus awamori were performed using wheat bran. The results are compared with previous work on synthetic medium. Major differences are that the wheat bran was not consumed completely as glucose or sucrose and that fungi growing on the wheat bran particles (adhesion growth) may occur depending on the inocula used. If the spore concentration was higher than 1.3 × 10 5 spores ml −1, adhesion growth dominated. If the concentration was lower than 1.8 × 10 4 spores ml −1, wheat bran free pellets and clean wheat bran particles dominated. In between these two values, the broth suspension consisted of wheat bran free pellets, clean wheat bran particles, and adhesion colonies. Solid substrate suppressed the growth in free filamentous mycelial form. A method was developed to separate the wheat bran particles from the pellets. The kinetics with complex medium appeared to be essentially the same as with synthetic medium. The ratio of biomass production to the sum of biomass production and CO 2 production was a function of time and independent of operation conditions, morphology, and substrates (sucrose, glucose, or wheat bran). Gas-liquid mass transfer coefficients during the fermentations were determined and compared with a correlation from the literature.

Influence of fermentation conditions and scale on the submerged fermentation of Aspergillus awamori

Aspergillus awamori is an interesting fungus for the industrial production of enzymes. To generate essential information, fermentations with this organism (CBS 115.52) were performed under various conditions. The ratio of the biomass production to the sum of biomass production and carbon dioxide production in time is used for deriving the yield and maintenance coefficient. This ratio can be used to estimate the biomass concentration from the CO 2 production on line as well. Pellets were the dominant morphology for most of the fermentations performed. The hairy length of the pellets first increased with time and then decreased. At a comparable specific energy dissipation rate, in the larger scale fermenters, pellets had a larger hairy length than in the smaller. The fraction of pellet biomass in the total biomass decreased with time at the beginning of the fermentations (0–20 h) and then reached a more or less stable value. Both counting the number of pellets during the fermentations and observing pellets under agitated but nongrowth conditions indicated that formation and breakage of pellets hardly occurred under the studied conditions.

Metabolic engineering: techniques for analysis of targets for genetic manipulations.

Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated.