Janusz Szczodrak

Technology for conversion of lignocellulosic biomass to ethanol

Abstract Current trends in production of fuel ethanol from lignocellulosic materials are reviewed. Particular emphasis has been laid on the microbial synthesis of cellulases, enzymatic hydrolysis, pretreatment of lignocellulosics, and their simultaneous saccharification and fermentation to ethyl alcohol. Some pilot-scale plants producing alcohol from biomass are also presented.

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Kluyveromyces fragilis

Neutral b-galactosidase from Kluy˝eromyces fragilis was immobilized on silanized porous glass modified by glutaralde- . hyde binding, with retention of more than 90% of its activity. Marked shifts in optimum pH from 7.0 to 6.0 and . temperature from 358 Ct o 508C of the solid-phase enzyme were observed together with high catalytic activity and reasonable stability at wider pH and temperature ranges than those of the free enzyme. Highly efficient lactose .

INSOLUBLE GLUCANS SYNTHESIZED BY CARIOGENIC STREPTOCOCCI : A STRUCTURAL STUDY

Of the three cariogenic streptococci grown in four various culture media, the strain Streptococcus mutans 20381 was found to produce large amounts of extracellular glucosyltransferase and water‐insoluble, adhesive exopolysaccharide when grown in batch culture on brain‐heart infusion broth. Methylation and nuclear magnetic resonance analyses revealed that the insoluble polymers synthesized by the crude glucosyltransferase preparations were mixed‐linkage (1 → 3), (1 → 6)‐α‐D‐glucans (so‐called mutans) with a greater proportion of 1,3 to 1,6 linkages and major branch points of 3,6‐linked glucose. The percentage content of different types of linkages in glucans varied widely and depended on the strain of cariogenic bacteria used to produce glucosyltransferase, and on the kind of medium utilized to cultivate mutans streptococci. The potential application of insoluble glucan produced by mutans streptococci is discussed.

Immobilization of cellulase and d-xylanase complexes from Aspergillus terreus F-413 on controlled porosity glasses

The major components of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase (see 1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. The final preparations contained over 20 mg protein g−1 glass beads. The activity retained was 71.6–98.1% for cellulase complexes and 81–100% for d-xylanase complexes. The immobilization of the enzymes spread their optimum pH range. Cellulose and d-xylan were quantitatively hydrolysed by the immobilized enzymes. The major reaction products were identified as a d-glucose and d-xylose respectively.

Biological study on carboxymethylated (1→3)-α-D-glucans from fruiting bodies of Ganoderma lucidum.

Water-insoluble, alkali-soluble polysaccharides (ASPs) were isolated from three fruiting bodies of the macromycete fungus Ganoderma lucidum. The structure of ASPs was determined using composition analysis, methylation analysis, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The analysis of the biological activity of the carboxymethylated (CM) (1→3)-α-D-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects against a tumor cell line (human cervical carcinoma HeLa), and two normal human cell lines (colon myofibroblasts CCD-18Co and epithelial cells CCD 841 CoTr). The chemical and spectroscopic investigations indicated that the ASPs from G. lucidum were (1→3)-α-D-glucans. After carboxymethylation (1→3)-α-D-glucans were tested in the range of 25-250 μg/mL concentrations. All the tested CM-(1→3)-α-D-glucans decreased the cellular metabolism of tumor and normal cells after 24h of incubation. The CM-(1→3)-α-D-glucans had no toxic effects on cervical carcinoma cells but reduced the viability of normal cells. The cytotoxic activity of the CM-(1→3)-α-D-glucans was concentration- and cell-type-dependent with normal cells more sensitive to their action than tumor cells. Generally, the CM-(1→3)-α-D-glucans tested did not have a free radical scavenging effect. It was concluded that the carboxymethylated derivatives of (1→3)-α-D-glucans isolated from the G. lucidum fruiting bodies are biologically active and after further detailed studies may be regarded as a dietary or therapeutic supplements.

(1 → 3)-α-d-Glucan hydrolases in dental biofilm prevention and control: A review

Dental plaque is a highly diverse biofilm, which has an important function in maintenance of oral and systemic health but in some conditions becomes a cause of oral diseases. In addition to mechanical plaque removal, current methods of dental plaque control involve the use of chemical agents against biofilm pathogens, which however, given the complexity of the oral microbiome, is not sufficiently effective. Hence, there is a need for development of new anti-biofilm approaches. Polysaccharides, especially (1→3),(1→6)-α-D-glucans, which are key structural and functional constituents of the biofilm matrix, seem to be a good target for future therapeutic strategies. In this review, we have focused on (1→3)-α-glucanases, which can limit the cariogenic properties of the dental plaque extracellular polysaccharides. These enzymes are not widely known and have not been exhaustively described in literature.

Hydrolysis of dextran by Penicillium notatum dextranase and identification of final digestion products

The basic hydrolysis parameters, i.e. molecular weight of substrate, reaction time, pH, temperature, and enzyme and substrate concentration, were standardized to maximize sugar yield from dextran. Thus, a 74% conversion of 4% dextran to sugar syrup was reached in 12 h using 6 dextranase units g − hydrolysed substrate. The high concentrations of sucrose in the reaction medium did not affect the hydrolysis eaeciency nor the activity of Penicillium notatum dextranase. Action patterns of dextranase from P. notatum on dextran were investigated. Analysis of the dextran hydrolysis end products indicated that P. notatum dextranase was a typical endo-enzyme, and isomaltose and isomaltotriose were identified as the primary final products of dextran hydrolysis.

Chemical characterization of a water insoluble (1 → 3)-α-d-glucan from an alkaline extract of Aspergillus wentii

The chemical structure of a water insoluble α-glucan isolated from the cell wall of Aspergillus wentii was described on the basis of total acid hydrolysis, methylation analysis, and 1D and 2D NMR studies (TOCSY, DQF-COSY, NOESY and HSQC) as well as other instrumental techniques. It was established that the analyzed preparation contained a linear polymer composed almost exclusively of (1→3)-linked α-d-glucose, with a molecular mass of about 850kDa. The polymer was divided into subunits separated by a short spacers of (1→4)-linked α-d-glucoses. Each subunit contained about 200 glucose residues.

Production and use of mutanase from Trichoderma harzianum for effective degradation of streptococcal mutans

Basic cultural parameters affecting mutanase production by Trichoderma harzianum F-340 in shaken flasks and aerated fermenter cultures have been standardized. The best medium for enzyme production was Mandels medium A with initial pH 5.3, supplemented with 0.3% mutan and 0.05% peptone and inoculated with 20% of the 72-h mycelium as inoculum. It was shown that mycelial mass, used in the culture medium as a sole carbon source, induced mutanase synthesis and could be utilized as an inexpensive and easily available substitute for bacterial mutan. Application of optimized medium and cultural conditions enabled us to obtain a high mutanase yield (0.6-0.7 U/mL, 2.0-2.5 U/mg protein) in a short period of time (3-5 days), which was much higher than the best reported in literature. The enzyme in crude state was stable in the pH range of 4.5-6.0, and at temperatures of up to 40oC; its maximum activity was recorded at 45oC and at pH 5.5. The mutanase preparation obtained from the T. harzianum fungus was relatively stable under storage conditions, and showed a high hydrolytic potential in reaction with a mixed-linkage (a-1,3, a-1,6) water-insoluble mutan of streptococcal origin (hydrolysis yield reached a value of 69% in 24 h). Steady-state measurement of the enzymic reaction products during the hydrolysis revealed that mutanase exhibited an exo type of action on mutan. Thin-layer chromatographic analysis showed that glucose was the primary final product of mutan hydrolysis with mutanase. The potential application of mutanase in dentistry is discussed.

Purification and characterization of an extracellular mutanase from Trichoderma harzianum

Trichoderma harzianum (CCM F-470) was found to produce large amounts of extracellular mutanase (0.33 U ml−1, 1.85 U mg protein−1) when grown aerobically on the optimized mutan medium in fermenter culture with an automatic pH control. The mutanase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The enzyme was recovered with a 94-fold increase in specific activity and a yield of 73%. The molecular weight of the purified enzyme proved to be 67 kDa, as estimated by SDS-PAGE, and to be 274 kDa, as determined by size-exclusion HPLC. These results indicate that the native mutanase is probably a tetramer protein. The isoelectric point was at 7.11, and the carbohydrate content in the purified enzyme was 4.42%. The pH and temperature optima were 5.5 and 40°C, respectively. The enzyme remained stable over a pH range of 4.5-6.0 and up to 35° for 1 h. The values of Km and Vmax under standard assay conditions were 1.2 × 10−3 g ml−1 and 8.48 × 10−2 U mg−1, respectively.