Sven Pedersen

Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates.

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsværd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo‐1,4‐β‐glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose‐releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid‐impregnated, steam‐exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.

Efficiency of New Fungal Cellulase Systems in Boosting Enzymatic Degradation of Barley Straw Lignocellulose

This study examined the cellulytic effects on steam‐pretreated barley straw of cellulose‐degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a β‐glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5–6.5) and temperature (35–65 °C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam‐pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.

Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant “Minimal” Enzyme Cocktail Containing β‐Xylosidase and Novel endo‐1,4‐β‐Xylanase and α‐l‐Arabinofuranosidase Activities

This study describes the identification of the key enzyme activities required in a “minimal” enzyme cocktail able to catalyze hydrolysis of water‐soluble and water‐insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose‐releasing and xylan‐depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel α‐l‐arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water‐soluble and water‐insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 α‐l‐arabinofuranosidase from H. insolens (Abf II), the GH51 α‐l‐arabinofuranosidase from M. giganteus (Abf III), a GH10 endo‐1,4‐β‐xylanase from H. insolens (Xyl III), and a GH3 β‐xylosidase from Trichoderma reesei (β‐xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water‐soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water‐insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 °C. A 10%:40%:50% mixture of Abf II, Abf III, and β‐xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 °C. The optimal dosages of the “minimal” enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water‐soluble wheat arabinoxylan, the water‐insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were ∼14, ∼18, and ∼27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei.

Next-Generation Catalysis for Renewables: Combining Enzymatic with Inorganic Heterogeneous Catalysis for Bulk Chemical Production

Nowadays, production of bulk and commodity chemicals from renewable feedstocks is widely debated and investigated as an alternative to the fossil platform. The conversion of biomass necessitates the development of a new generation of catalysts that enable new kinds of reactions from a different chemical platform under different conditions than those conventionally employed. Indeed, new process and catalyst concepts need to be established. Both enzymatic catalysis (biocatalysis) and heterogeneous inorganic catalysis are likely to play a major role and, potentially, be combined. One type of combination involves one‐pot cascade catalysis with active sites from bio‐ and inorganic catalysts. In this article the emphasis is placed specifically on oxidase systems involving the coproduction of hydrogen peroxide, which can be used to create new in situ collaborative oxidation reactions for bulk chemical production.

Development of new α-amylases for raw starch hydrolysis

This paper describes the discovery of a new 4 domain α-amylase from Anoxybacillus contaminans which very efficiently hydrolyses raw starch granules. Compared to traditional starch liquefying α-amylases, this new 4 domain α-amylase contains a starch binding domain. The presence of this starch-binding domain enables the enzyme to efficiently hydrolyse starch at a temperature below the gelatinisation temperature. At a reaction temperature of 60°C and in combination with a glucoamylase from Aspergillusniger it was possible to liquefy 99% of the starch obtaining a DX value of 95%. Furthermore, we describe how the current HFCS process can be turned into a low temperature simultaneous liquefaction and saccharification process by using this new 4 domain α-amylase in combination with a glucoamylase.

Chemoenzymatic Combination of Glucose Oxidase with Titanium Silicalite‐1

The use and integration of heterogeneous chemical catalysis and biocatalysis, especially using enzymes, will become increasingly important in a future chemical industry based on renewable resources. A range of alternative renewable chemical platforms have already been proposed from biomass, and compared to the conventional fossil-based platform these are radically different. The main difference lies in the high degree of oxyfunctionality of sugar molecules and (poly)alcohols compared to the low functionality of hydrocarbons and aromatics derived from crude oil. Consequently, the approaches for converting these alternative platforms are not identical. In conventional oil-based chemistry, functionality is introduced, whereas functionality should either be removed or used selectively when working with a renewable platform. Furthermore, biomass is usually in the solid form and the inherent oxyfunctionality renders it difficult, if not impossible, to use existing processes to convert the renewable platform into useful chemicals, because it is not easily handled in the gas phase. Therefore, the conversion of biomass will necessarily be carried out in the liquid (aqueous) phase. For the aforementioned reasons, there is now a need to develop a new generation of catalysts, which are able to facilitate the conversion of especially biomass-derived resources in the next generation of chemical processes. It will therefore be advantageous to combine the high stereo-, regio-, and chemoselectivity of enzymes with the diversity, tolerance, and ability of chemical catalysts to operate under various conditions and transform various substrates. In this way the advantages of each catalyst system may be exploited. 5] Additionally these advantages can be integrated and in some cases combined in heterogeneous one-pot reactions with enzymes or even using enzymes linked to the chemical catalyst to further intensify the process and to facilitate catalyst recycling and separation. One such system is the combination of oxidase enzymes with hydrogen peroxide-active titanium silicalite-1 (TS-1—a redox-active molecular sieve with MFI framework). Oxidases (E.C. 1.1.3) provide a useful catalytic approach to selective oxidation under mild conditions using air as a safe oxidant. They are probably more useful than oxygenases, given the need for expensive cofactors, which need to be recycled, when using the latter. This suggested combination has several advantages:

Effect of Enzymatic Treatment of Different Starch Sources on the in Vitro Rate and Extent of Starch Digestion

Gelatinized wheat, potato and waxy maize starches were treated enzymatically in order to increase the degree of branching of the amylopectin fraction and thereby change the starch degradation profile towards a higher proportion of slowly digestible starch (SDS). The materials were characterized by single-pulse 1H HR-MAS NMR spectroscopy and in vitro digestion profile according to the Englyst procedure. Using various concentrations and incubation times with branching enzyme (EC 2.4.1.18) without or with additional treatment with the hydrolytic enzymes; β-amylase (EC 3.2.1.2), α-glucosidase (EC 3.2.1.20), or amyloglucosidase (EC 3.2.1.3) the proportion of α-(1–6) linkages was increased by up to a factor of 4.1, 5 and 5.8 in waxy maize, wheat and potato starches, respectively. The proportion of SDS was significantly increased when using hydrolytic enzymes after treatment with branching enzyme but it was only for waxy maize that the proportion of α-(1–6) bonds and the in vitro digestion profile was significantly correlated.

Characterization of oligosaccharides from industrial fermentation residues by matrix-assisted laser desorption/ionization, electro spray mass spectrometry, and gas chromatography mass spectrometry

We report here the preliminary characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ion trap mass spectrometry (ESI-ITMS), and gas chromatography mass spectrometry (GC-MS). After sample cleaning with carbon graphite columns, analysis of oligosaccharides present in the sample using MALDI-TOF-MS resulted in identification of molecular ions representing sodiated hexose and pentose oligo/polysaccharides. The GC-MS analyses revealed that the signals observed in the mass spectrum for hexose oligomers represent linear structures, whereas the pentose oligomers were identified as arabinoxylans with a (1»4) linked Xylp backbone where the Xylp residues were either not substituted or singly substituted with Araf branching residues at positions C-2 or C-3 of the Xylp ring. Analyses by ESI-ITMS of the signals corresponding to arabinoxylan oligosaccharides with four and five monosaccharide residues showed the presence of isomeric structure differing in degree of branching and localization of the branched residue along the Xylp backbone.

Resistant Starch but Not Enzymatically Modified Waxy Maize Delays Development of Diabetes in Zucker Diabetic Fatty Rats

Background: The incidence of type 2 diabetes (T2D) is increasing worldwide, and nutritional management of circulating glucose may be a strategic tool in the prevention of T2D.Objective: We studied whether enzymatically modified waxy maize with an increased degree of branching delayed the onset of diabetes in male Zucker diabetic fatty (ZDF) rats.Methods: Forty-eight male ZDF rats, aged 5 wk, were divided into 4 groups and fed experimental diets for 9 wk that contained 52.95% starch: gelatinized corn starch (S), glucidex (GLU), resistant starch (RS), or enzymatically modified starch (EMS). Blood glucose after feed deprivation was assessed every second week; blood samples taken at run-in and at the end of the experiment were analyzed for glycated hemoglobin (HbA1c) and plasma glucose, insulin, and lipids. During weeks 2 and 8, urine was collected for metabolomic analysis.Results: Based on blood glucose concentrations in feed-deprived rats, none of the groups developed diabetes. However, in week 9, plasma glucose after feed deprivation was significantly lower in rats fed the S and RS diets (13.5 mmol/L) than in rats fed the GLU and EMS diets (17.0-18.9 mmol/L), and rats fed RS had lower HbA1c (4.9%) than rats fed the S, GLU, and EMS (5.6-6.1%) diets. The homeostasis model assessment of insulin resistance was significantly lower in rats fed RS than in rats fed the other diets (185 compared with 311-360), indicating that rats fed the S, GLU, and EMS diets were diabetic, and a 100% higher urine excretion during week 8 in rats fed the GLU and EMS diets than that of rats fed S and RS showed that they were diabetic. Urinary nontargeted metabolomics revealed that the diabetic state of rats fed S, GLU, and EMS diets influenced microbial metabolism, as well as amino acid, lipid, and vitamin metabolism.Conclusions: EMS did not delay the onset of diabetes in ZDF rats, whereas rats fed RS showed no signs of diabetes.

Hydration properties and phosphorous speciation in native, gelatinized and enzymatically modified potato starch analyzed by solid-state MAS NMR

Hydration of granular, gelatinized and molecularly modified states of potato starch in terms of molecular mobility were analyzed by (13)C and (31)P solid-state MAS NMR. Gelatinization (GEL) tremendously reduced the immobile fraction compared to native (NA) starch granules. This effect was enhanced by enzyme-assisted catalytic branching with branching enzyme (BE) or combined BE and β-amylase (BB) catalyzed exo-hydrolysis. Carbons of the glycosidic α-1,6 linkages required high hydration rates before adopting uniform chemical shifts indicating solid-state disorder and poor water accessibility. Comparative analysis of wheat and waxy maize starches demonstrated that starches were similar upon gelatinization independent of botanical origin and that the torsion angles of the glycosidic linkages were averages of the crystalline A and B type structures. In starch suspension phosphorous in immobile regions was only observed in NA starch. Moreover phosphorous was observed in a minor pH-insensitive form and as major phosphate in hydrated GEL and BE starches.