George Szakacs

Background: Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Results: Lignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures. Conclusion: Enzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional β-xylosidase activity.

To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates. Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50 degrees C. Molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size ( approximately 37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities. Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase. Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.

To screen various Streptomyces cultures producing L-leucine aminopeptidase (LAP).Twenty-one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B-3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8.0-8.5 and the temperature optima varied between 50 and 65 degrees C. LAP of S. mobaraensis was stable at 60 degrees C and pH 8.5 for 60 min. Metal ion salts, CoCl(2).6H(2)O and ZnSO(4).7H(2)O in 0.7 mmol l(-1) concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa.Streptomyces mobaraensis NRRL B-3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable.A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N-terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.

Among numerous mesophilic fungi screened for sterol esterase activity followed by the esterification reaction between plant β‐sitosterol and lauric acid in organic solvent, six Aspergillus strains were selected as the most active producers. These fungi had not been studied previously for sterol esterase production. The fungi were cultivated under solid state fermentation (SSF) conditions. The gently dried SSF cultures as such were tested in the esterification reactions, without any special enzyme isolation and purification (downstream) processes. All the six Aspergillus SSF preparations were able to synthesize sterol esters. Sterol esterase activity of these GRAS cleared Aspergillus strains was inducible by sterol ester supplementation to the SSF medium and showed remarkably different moisture optimum during growth as compared to the production of lipase (determined by pNP‐palmitate). Genome analysis revealed that sterol esterase production might be a common feature of many Aspergillus species. The synthetic usefulness of the best SSF preparations of A. oryzae NRRL 6270 and A. sojae NRRL 6271 was demonstrated by synthesis of esters of plant sterols with lauric acid resulting in 45–63% conversions (GC) and 27–38% isolated yields of steryl laurates. The isomer preference of A. oryzae NRRL 6270 towards the 10E,12Z isomer of conjugated linoleic acid (CLA) in the esterification reaction with plant sterols was also determined.

Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central and South America, including the Caribbean Islands, and southwestern and southeastern Asia. Morphological and physiological characters, the internal transcribed spacer regions of the rDNA cluster (ITS1-5.8SrDNA-ITS2) and partial sequences of translation elongation factor 1-alpha (tef1) are described. Trichoderma brevicompactum is characterized by a pachybasium-type morphology, morphologically resembling other small-spored species referable to Trichoderma section Pachybasium but with essentially subglobose conidia. It is most closely related phylogenetically to Hypocrea lutea, from which it differs in morphological and physiological characters.

Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase, and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.

Fourteen thermophilic filamentous fungi were cultivated on two shake flask media and the supernatants were assayed for lipase/carboxylesterase activities using olive‐oil, p‐nitrophenyl palmitate and p‐nitrophenyl butyrate as substrates. The crude enzyme powders (acetone precipitated supernatants) were tested as biocatalysts in organic solvents. Kinetic resolution of racemic 1‐phenylethanol (rac‐1) and desymmetrisation of 2‐acyloxypropan‐1,3‐diols (3a, b) by acetylation with vinyl acetate were chosen for testing the biocatalytic abilities of these preparations. The tested biocatalysts proved to be comparable to the commercially available enzymes with respect to the degree of enantiomer selectivity, whereas they exhibited a wider range of enantiotopic selectivity than the most common commercial enzymes.