Matti Leisola

A rare sugar xylitol. Part II: biotechnological production and future applications of xylitol

Xylitol is the first rare sugar that has global markets. It has beneficial health properties and represents an alternative to current conventional sweeteners. Industrially, xylitol is produced by chemical hydrogenation of d-xylose into xylitol. The biotechnological method of producing xylitol by metabolically engineered yeasts, Saccharomyces cerevisiae or Candida, has been studied as an alternative to the chemical method. Due to the industrial scale of production, xylitol serves as an inexpensive starting material for the production of other rare sugars. The second part of this mini-review on xylitol will look more closely at the biotechnological production and future applications of the rare sugar, xylitol.

Comparison of ligninolytic activities of selected white-rot fungi

SummarySix fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.

Transglycosylation products of cellulase system ofTrichoderma reesei

SummaryFrom cellulose and cellobiose the formation of sophorose, laminaribiose, and gentiobiose was catalyzed byTrichoderma reesei culture filtrate containing exo- and endoglucanase and β-glucosidase activity and from cellobiose by a broken cell suspension fromT.reesei with β-glucosidase activity. The results indicate that β-glucosidase is the component responsible for transglycosylation reaction catalyzed byT.reesei cellulase enzyme complex.

A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism

Thermal stability and other functional properties of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII; family 11) were studied by designed mutations. Mutations at three positions were introduced to the XYNII mutant containing a disulfide bridge (S110C-N154C) in the alpha-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65 degrees C. An additional mutation at the C-terminus of the alpha-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55 degrees C but not at 65 degrees C. The mutations N11D and N38E increased the half-life to about 100 min. Due to the stabilizing mutations the pH stability increased in a wide pH range, but at the same time the activity decreased both in acidic and neutral-alkaline pH, the pH optimum being at pH region 5-6. There was no essential difference between the specific activities of the mutants and the wild-type XYNII.

Metabolic Engineering of Lactobacillus helveticus CNRZ32 for Production of Pure l-(+)-Lactic Acid

ABSTRACT Expression of d-(−)-lactate dehydrogenase (d-LDH) and l-(+)-LDH genes (ldhDand ldhL, respectively) and production ofd-(−)- and l-(+)-lactic acid were studied inLactobacillus helveticus CNRZ32. In order to develop a host for production of pure l-(+)-isomer of lactic acid, twoldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene ofldhD with an additional copy of the structural gene (ldhL) of l-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The twod-LDH-negative strains produced onlyl-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum l-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables forl-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41°C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.

A rare sugar xylitol. Part I: the biochemistry and biosynthesis of xylitol

The rare sugar xylitol is a five-carbon polyol (pentitol) that has beneficial health effects. Xylitol has global markets and, therefore, it represents an alternative to current dominant sweeteners. The research on microbial reduction of d-xylose to xylitol has been focused on metabolically engineered Saccharomycess cerevisiae and Candida strains. The Candida strains have an advantage over the metabolically engineered S. cerevisiae in terms of d-xylose uptake and maintenance of the intracellular redox balance. Due to the current industrial scale production of xylitol, it has become an inexpensive starting material for the production of other rare sugar. The first part of this mini-review concentrates on the biochemistry of xylitol biosynthesis and the problems related to intracellular redox balance.

Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30

Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was cultivated using polysaccharides (cellulose or xylan) as the main carbon sources, we now report a slightly lower pH value for maximal xylanase levels. The highest xylanase activity (IU/ml) on the lactose-based medium was observed at pH 6.0 compared to pH 7.0 on the polysaccharide-based media. When the pattern of different xylanases was analyzed by isoelectric focusing and activity zymogram, we observed that a low pH (4.0) favoured the production of xylanase I, whilst a high pH (6.0) favoured the production of xylanase III. Xylanase II was clearly produced at both pH values. The results at pH 4 and 6 correlate with the pH activity profiles of xylanase I, II and III. Hence, the different T. reesei xylanases were produced according to which enzyme is most active in that particular environment.

High-level production of D-mannitol with membrane cell-recycle bioreactor.

Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state. The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures. High volumetric mannitol productivity (26.2 g l−1 h−1) and mannitol yield (97 mol%) were achieved. Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches. Applying response surface methodology, the temperature and pH were studied with respect to specific mannitol productivity and yield. Moreover, increasing the initial fructose concentration from 100 to 120 and 140 g l−1 resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase (MDH). Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters. Journal of Industrial Microbiology & Biotechnology (2002) 29, 44–49 doi:10.1038/sj.jim.7000262

Protein engineering: opportunities and challenges

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.

Optimization of enzymatic transesterification of rapeseed oil ester using response surface and principal component methodology

Statistical experimental design combined with principal component analysis was used to evaluate interdependence of process variables in enzymatic transesterification. The reaction studied was alcoholysis of rapeseed oil methyl ester (biodiesel) and trimethylolpropane. The reaction products are trimethylolpropane esters which can be used as raw materials for biodegradable hydraulic fluid and other lubricants. The methods used revealed some hidden factors that affect the reaction. Water activity and elimination of the by-product methanol were found to be the principal factors affecting the product yield. Mono-, di-, and trisubstituted esters were produced in different optimum conditions. The model developed for monosubstituted ester was verified with a bubble column experiment.