Klaus D. Kulbe

Production of fungal xylanases

Applications of xylanases can be found in the food, feed and pulp/paper industry. Filamentous fungi are particularly interesting producers of this enzyme from an industrial point of view, due to the fact that they excrete xylanases into the medium. Furthermore, xylanase levels from fungal cultures are generally much higher than those from yeasts or bacteria. In addition to xylanase, fungi typically produce several accessory xylanolytic enzymes, which are necessary for debranching substituted xylans. An important factor for efficient xylanase production is the choice of an appropriate inducing substrate, either insoluble or soluble, as well as optimization of the medium composition. In addition, the substrate can influence the concomitant formation of cellulolytic enzymes in certain organisms. Bioprocess parameters, that can affect activities and productivities of xylanase attained in a fermentation process, as well as the concurrent formation of cellulases, include the pH, temperature, or agitation. Xylanase activities produced by different organisms, including filamentous fungi and yeasts, are compared for both submerged and solid-state fermentations. When available, data on the concurrent formation of cellulolytic enzyme activities are included. On an industrial scale, xylanases are produced mainly by Aspergillus and Trichoderma spp. A list of commercially available xylanases and their potential applications is given.

Influence of a synbiotic mixture consisting of Lactobacillus acidophilus 74-2 and a fructooligosaccharide preparation on the microbial ecology sustained in a simulation of the human intestinal microbial ecosystem (SHIME reactor)

Abstract Lactobacillus acidophilus 74-2, which is used in probiotic products, was administered, with fructooligosaccharide in a milk-based product, to the second vessel (duodenum/jejunum) of the SHIME reactor, an in vitro simulation of the human intestinal microbial ecology. The main focus of this study was to monitor the changes of the population density of selected bacterial species in the intestine and the changes of metabolic activities during the supplementation of L.acidophilus and fructooligosaccharide in the SHIME reactor. Interestingly, the addition of L. acidophilus 74-2 with fructooligosaccharide gave rise to an increase of bifidobacteria. Moreover, major positive changes occurred in the production of volatile fatty acids: a strong upward trend was observed especially in the case of butyric acid and propionic acid. Furthermore a noticeable increase of β-galactosidase activity was monitored, while the activity of β-glucuronidase, generally considered undesirable, declined.

Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor

We have developed a new process for the production of xylitol from D‐xylose by enzyme technology. An NADH‐dependent xylose reductase (XR) from Candida tenuis catalyzes the reduction of xylose, which is coupled to enzymatic oxidations of D‐glucose or D‐xylose by glucose dehydrogenase from Bacillus cereus to make achievable an up to 10,000‐fold regeneration of NADH per cycle of discontinuous conversion. Using a simple kinetic model as a tool for process optimization, suitable conditions with regard to performance and stability of the multi‐component reaction system were established, and 300 g/L of substrate could be converted in yields above 96% in one single batch reaction. Due to selective and over 98% complete retention of the native coenzyme by negatively charged nanofiltration membranes used in a continuously operated enzyme reactor, a specific productivity of 80 g xylitol per liter, day, and kilounit of XR was maintained over the 150‐h reaction time with only a single dosage of NADH.

A simple assay for measuring cellobiose dehydrogenase activity in the presence of laccase.

The commonly used assay for measuring cellobiose dehydrogenase (CDH) activity, based on the reduction of dichlorophenol-indophenol (DCIP), has been adapted to measure this enzyme activity in the presence of laccase, which is often formed concurrently with CDH by a number of fungi. Laccase interferes with the assay by rapidly reoxidizing the reduced form of DCIP and can mask CDH activity completely. It can be conveniently and completely inhibited by 4 mM fluoride in the assay, while CDH activity is only slightly affected by the addition of this inhibitor. The modified assay enables the detection of low CDH activities even in the presence of very high excesses of laccase. It should be useful for screening culture supernatants of wood-degrading fungi for CDH since the assay is rapid and uses inexpensive and nontoxic reagents. Furthermore, it might be used for the detection of other enzyme activities which are assayed by following the reduction of quinones or analogue compounds such as DCIP.

Continuous enzymatic regeneration of redox mediators used in biotransformation reactions employing flavoproteins

Abstract Oxidoreductases are a group of enzymes that have been regarded uneconomical for industrial processes due to their dependence on cofactors or prosthetic groups for activity and the difficulties of regenerating these. Especially, flavoproteins have long been neglected for biocatalytical applications. The prosthetic group of some of these enzymes, but not all, can be regenerated by oxygen, resulting in hydrogen peroxide formation, which is detrimental to enzyme stability. As a contribution to alleviating this problem, a novel concept for the regeneration of electron acceptors (redox mediators) for flavoenzymes is described. Flavin-containing enzymes such as cellobiose dehydrogenase (CDH) or pyranose oxidase (P2O) are used in conjunction with laccases and a redox mediator. The flavin of the synthetic enzyme is reduced while the oxidized product of interest is formed, in turn, the flavin is reoxidized with the help of an electron acceptor, which then is regenerated using a laccase. Laccases are copper containing phenol oxidases that can transfer four electrons to oxygen, producing two molecules of water. Preliminary screening experiments with different redox mediators, and a coupled enzyme system of CDH and laccase, showed that a wide variety of different substances can efficiently shuttle electrons between these two enzymes. Among them are substituted and unsubstituted ortho - and para -quinones, benzoquinone imines, cation radicals such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), redox dyes such as phenothiazines or phenoxazines, as well as iron complexes. Experiments in which CDH completely oxidizes lactose to lactobionic acid and P2O entirely converts glucose to 2-keto-glucose are presented. Catalytic amounts of redox mediators are used and continuously regenerated by a laccase. Specific productivities of up to 19.3 g·(h·kU) −1 and 72 g·(h·kU) −1 for CDH and P2O, respectively, were found. The total turnover numbers (TTNs) for the two enzymes used were in the range of 10 5 –10 6 . Oxygen supply for the laccase is a crucial factor in avoiding rate limitation. Undeniably, this system facilitates the efficient use of a hitherto underexploited group of enzymes for preparative purposes.

Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose. I. The properties of two thermostable β-glycosidases

Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with SsbetaGly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but not that of SsbetaGly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than SsbetaGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki approximately 0.3 M), and at 80 degrees C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsbetaGly, respectively. SsbetaGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsbetaGly, compared with CelB. Overall, CelB and SsbetaGly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at >/=70 degrees C.

Enhanced Formation of Extracellular Laccase Activity by the White-Rot Fungus Trametes multicolor

The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The formation of extracellular laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5-1 mM, which were added during the growth phase of the fungus. Other medium components important for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO4 x 7H2O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory fermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.

Continuous Enzymatic Regeneration of Electron Acceptors Used by Flavoenzymes: Cellobiose Dehydrogenase-Catalyzed Production of Lactobionic Acid as an Example

An efficient enzymatic bioprocess is described in which lactose, an abundant renewable resource produced by the dairy industry, is completely and efficiently converted with a specific productivity of up to 32 g (kU h)−1 into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the fungal enzyme cellobiose dehydrogenase which oxidizes several β-1,4-linked disaccharides including lactose specifically at position C-1 of the reducing sugar moiety to the corresponding lactones. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H2O-producing, copper-containing oxidase, and therefore has to be added in low, catalytic amounts only. Redox mediators that were successfully employed in this novel process and hence are compatible with the laccase regeneration system include benzoquinone, ABTS, ferricyanide, or ferrocene, amongst others. Factors affecting operational stability of the biocatalysts employed in this process include the redox mediator used, the temperature, and importantly the volumetric gas flow necessary for maintaining the dissolved oxygen tension. Lactobionic acid is a mild and sweet tasting acid with excellent chelating properties. These useful characteristics have lead to a growing number of patents for diverse applications in the food, pharmaceutical and detergent industries.

Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable β-glycosidases

During lactose conversion at 70 degrees C, when catalyzed by beta-glycosidases from the archea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB), galactosyl transfer to acceptors other than water competes efficiently with complete hydrolysis of substrate. This process leads to transient formation of a range of new products, mainly disaccharides and trisaccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chromatography. At 270 g/L initial lactose, they accumulate at a maximum concentration of 86 g/L at 80% lactose conversion. With both enzymes, the molar ratio of trisaccharides to disaccharides is maximal at an early stage of reaction and decreases directly proportional to increasing substrate conversion. Overall, CelB produces about 6% more hydrolysis byproducts than SsbetaGly. However, the product spectrum of SsbetaGly is richer in trisaccharides, and this agrees with results obtained from the steady-state kinetics analyses of galactosyl transfer catalyzed by SsbetaGly and CelB. The major transgalactosylation products of SsbetaGly and CelB have been identified. They are beta-D-Galp-(1-->3)-Glc and beta-D-Galp-(1-->6)-Glc, and beta-D-Galp-(1-->3)-lactose and beta-D-Galp-(1-->6)-lactose, and their formation and degradation have been shown to be dependent upon lactose conversion. Both enzymes accumulate beta(1-->6)-linked glycosides, particularly allolactose, at a late stage of reaction. Because a high oligosaccharide concentration prevails until about 80% lactose conversion, thermostable beta-glycosidases are efficient for oligosaccharide production from lactose. Therefore, they prove to be stable and versatile catalysts for lactose utilization.

Continuous enzymatic production of lactobionic acid using glucose-fructose oxidoreductase in an ultrafiltration membrane reactor

Glucose-fructose oxidoreductase from Zymomonas mobilis catalyzed the oxidation of various aldose sugars to the corresponding aldonic acids. The enzyme was used for the selective and high-yield conversion of lactose to lactobionic acid in batch, fed-batch and continous reaction mode. A productivity of 110 g L d was obtained in an ultrafiltration membrane reactor, operated for 70 h.