Ossi Pastinen

Novel reactions of xylose isomerase from Streptomyces rubiginosus

Abstract Three novel substrates— d -arabinose, l -ribose, and d -lyxose—are described for the industrially important xylose isomerase (glucose isomerase). Furthermore, it was shown that contrary to previous studies, two different reaction products are formed when pentose sugars are used as substrates. In extended reactions an apparent equilibrium is formed between both d - and l -forms of arabinose, ribulose, and ribose as well as d -xylose, d -lyxose, and d -xylulose. The new reactions are discussed in the light of previously described reaction mechanisms for this enzyme.

Stability of native and cross-linked crystalline glucose isomerase.

Stabilities of native and cross-linked crystalline forms of Streptomyces rubiginosus glucose isomerase were compared in buffer and in 45% glucose/fructose solutions. The cross-linked crystalline form of the enzyme was more stable in the presence of substrate while in a buffer solution the native enzyme was more stable. Inactivation of native enzyme in buffer did not obey first-order kinetics but proceeded with a rapid first phase followed by a stable phase. This stabilization is interpreted to be a result of a conformational change in the protein structure. Inactivation of the native enzyme in buffer was directly related to protein precipitation. In the presence of high substrate concentration, the inactivation was related to browning reactions between the enzyme and the reactive sugar, resulting in soluble sugar-protein complexes.

Enzymatic saccharification of pretreated wheat straw: Comparison of solids-recycling, sequential hydrolysis and batch hydrolysis

In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series.

Xylitol purification by cross-linked glucose isomerase crystals

Xylitol is specifically bound by active, cross-linked glucose isomerase crystals (CLGI). CLGI can be used to purify xylitol or concentrate it from dilute and impure solutions. Bound xylitol can be eluted from CLGI by Ca2 and the material reactivated by Mg2. The binding capacity is 1 mg xylitol per 525 mg CLGI which equals one molecule per active center. CLGI can further be used to purify xylitol and sorbitol from impure mixtures of arabinitol, mannitol, ribitol and monosaccharides.

Determination of surface-accessible acidic hydroxyls and surface area of lignin by cationic dye adsorption

A new colorimetric method for determining the surface-accessible acidic lignin hydroxyl groups in lignocellulose solid fractions was developed. The method is based on selective adsorption of Azure B, a basic dye, onto acidic hydroxyl groups of lignin. Selectivity of adsorption of Azure B on lignin was demonstrated using lignin and cellulose materials as adsorbents. Adsorption isotherms of Azure B on wheat straw (WS), sugarcane bagasse (SGB), oat husk, and isolated lignin materials were determined. The maximum adsorption capacities predicted by the Langmuir isotherms were used to calculate the amounts of surface-accessible acidic hydroxyl groups. WS contained 1.7-times more acidic hydroxyls (0.21 mmol/g) and higher surface area of lignin (84 m(2)/g) than SGB or oat husk materials. Equations for determining the amount of surface-accessible acidic hydroxyls in solid fractions of the three plant materials by a single point measurement were developed. A method for high-throughput characterization of lignocellulosic materials is now available.

Xylose isomerase catalysed novel hexose epimerization

Novel hexose substrates and new epimerization reactions catalysed by the industrially important D-xylose ketol-isomerase (E.C. 5. 3. 1. 5.) are described. Isomerization equilibrium betweer D-allose and D-psicose or D-sorbose and D-gulose is obtained starting from either of the substrates. D-altrose is formed in the mixture of D-allose and D-psicose as an epimerization product and D-idose in the mixture of D-sorbose and D-gulose.

Autohydrolysis and aqueous ammonia extraction of wheat straw: effect of treatment severity on yield and structure of hemicellulose and lignin

The objective of this study was to elucidate the impact of autohydrolysis severity on the yield and structure of wheat straw hemicellulose and lignin. The autohydrolysis treatments were carried out at maximum temperatures between 170 °C and 200 °C. The autohydrolysis liquors were separated and the solids were successively extracted with aqueous ammonia either in moderate or high intensity extraction conditions to dissolve lignin for analysis. Increasing autohydrolysis severity decreased the molar mass of the aqueous ammonia extracts from 5450 g mol−1 to 1810 g mol−1, and carbohydrate content from 6% to 0.1%. The optimum autohydrolysis severity (log R0 = 3.81) for xylan recovery released mainly oligomeric arabinoxylans at 66% xylan recovery yield. Drastic degradation of pentoses occurred beyond the optimum severity. As an indication of accumulation of “pseudo-lignin” during autohydrolysis, decreasing relative aromaticity in the aqueous ammonia extracts as a function of autohydrolysis severity was shown. The finding was confirmed by quantitative analysis of the cupric oxide oxidation products of lignin suggesting up to 55% decrease in the relative amount of native lignin at the highest severity. These results show the importance of distinguishing between lignin and “pseudo-lignin” in fractions obtained from lignocellulosic materials subjected to acidic pretreatment.

Cross-linked glucose isomerase crystals as a liquid chromatographic separation material

Cross-linked crystals of glucose isomerase (CLGI) were characterized as a liquid chromatographic separation material. The experiments were done with crystals having an average diameter of 83 µm. Porosity (epsilon(p)) and pore size distribution of the CLGI crystals were measured with size exclusion chromatography using D(2)O and polyethylene glycols as probes. CLGI material was capable of separating <1000 g/mol polyethylene glycols. Fifty two percent (epsilon(p) = 0.47) of the total crystal volume was in pores. Pore size measurement showed that CLGI crystals were microporous material, having an average apparent pore diameter of 29 +/- 0.08 Å. CLGI material separated n-alcohols C(1) to C(8) based on the hydrophobic interaction between the protein material and the carbon chain of the alcohols. Height equivalent to a theoretical plate (HETP, in millimeters) ranged from 1.6 to 0.89 for the C(1) to C(7) n-alcohol series. Despite the large crystal size, CLGI as a chirally active phase effectively separated D- and L-arabitol (R(s) = 0.58) and showed potential for chiral separation of amino acids.

Lignin—Designed Randomness

0 0 1 282 1608 Biologic Institute 13 3 1887 14.0 Normal 0 false false false EN-US ZH-CN X-NONE Humans have long used wood as a structural material for some of the same reasons that trees use it—it combines great strength, flexibility and durability with a relatively low density. These desirable properties depend partly on lignin , a major chemical constituent of many plants, including trees. Lignin is the most abundant aromatic polymer on earth and the second most abundant organic polymer of any kind, exceeded only by cellulose. It is estimated that 30% of the earth’s non-fossil organic carbon is in the form of lignin. Considering its massive abundance and its high energy content (40% higher than cellulose, gram for gram), it is striking that no organism seems to have tapped it as an energy source. After posing this as an evolutionary enigma, we prepare to address it by reviewing what is known about the structure, biosynthesis and biodegradation of wood in general and of lignin in particular. Then, returning to the enigma, we ask whether it is more readily explained within a Darwinian framework or a design framework. The Darwinian account must somehow reconcile 400 million years of failure to evolve a relatively modest innovation—growth on lignin—with a long list of spectacular innovations thought to have evolved in a fraction of that time. How can one mechanism have been at the same time so effective and so ineffective? That tension vanishes completely when the design perspective is adopted. Terrestrial animal life is crucially dependent on terrestrial plant life, which is crucially dependent on soil, which is crucially dependent on the gradual photo- and biodegradation of lignin. Fungi accomplish th e bio degradation, and the surprising fact that it costs them energy to do so keeps the process gradual. The peculiar properties of lignin therefore make perfect sense when seen as part of a coherent design for the entire ecosystem of our planet.

Simultaneous catalysis and product separation by cross-linked enzyme crystals

Crystalline cross-linked xylose isomerase (CLXI, EC 5.3.1.5) and xylanase (CLX, EC 3.2.1.8) were studied in a packed-bed reactor for simultaneous catalytic reaction and separation of substrates from reaction products. Streptomyces rubiginosus xylose isomerase catalyzed a slow isomerization of L-arabinose to L-ribulose and an epimerization to L-ribose. In equilibrium the reaction mixture contained 52.5% arabinose, 22.5% ribulose, and 25% ribose. In a packed-bed column filled with CLXI, a simultaneous reaction and separation resulted in fractions where arabinose concentration varied between 100-0%, ribulose between 0-55%, and ribose between 0-100%. Trichoderma reesei xylanase II hydrolyzed and transferred xylotetraose mainly to xylotriose and xylobiose. In a packed-bed column filled with CLX, xylotetraose rapidly reacted to xylobiose and xylose by a mechanism that is not yet fully understood.