Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.     

环境条件对双叉犀金龟成虫存活及繁殖的影响

双叉犀金龟Allomyrina dichotoma是一种重要的森林资源昆虫,本文研究了环境温度（20℃、23℃、26℃、29℃、32℃）及菌糠含水量（35%、45%、55%、65%）对双叉犀金龟成虫寿命及子代数量的影响。结果表明,双叉犀金龟雌雄虫寿命均随温度的升高呈显著缩短,20℃~26℃下雌虫寿命在55~62 d之间,显著长于29℃~32℃处理;20℃~23℃下的雄虫寿命在33~48 d之间,显著长于26℃~32℃处理;环境温度及菌糠含水量均对双叉犀金龟成虫子代数量有显著影响,26℃时,4个菌糠含水量处理的平均子代数量为31.42 头/雌,显著高于其它温度处理,菌糠含水量55%~65%时,5个温度处理的平均子代数量为18.83~19.12 头/雌,显著高于其它菌糠含水量处理,其中以温度26℃、菌糠含水量55%处理的双叉犀金龟子代数量最高,达45.42 头/雌。各温度下双叉犀金龟繁殖子代数量均随时间的延长呈先上升后下降的趋势,各温度下产卵繁殖的高峰时间存在明显差异。在26℃时,双叉犀金龟各时间段子代数量均高于其它温度,在35~45 d时,达到产卵高峰期（12.33 头/雌）,综合各指标,建议利用菌糠饲养双叉犀金龟时,其成虫最佳环境条件为26℃,菌糠含水量55%。     

Expanding the applicability of cytochrome P450s and other haemoproteins

Cytochrome P450BM3 has long been regarded as a promising candidate for use as a biocatalyst, owing to its excellent efficiency for the hydroxylation of unactivated C–H bonds. However, because of its high substrate specificity, its possible applications have been severely limited. Consequently, various approaches have been proposed to overcome the enzyme's natural limitations, thereby expanding its substrate scope to encompass non-native substrates, evoking chemoselectivity, regioselectivity and stereoselectivity and enabling previously inaccessible chemical conversions. Herein, these approaches will be classified into three categories: (1) mutagenesis including directed evolution, (2) haem substitution with artificial cofactors and (3) use of substrate mimics, ‘decoy molecules’. Herein, we highlight the representative work that has been conducted in above three categories for discussion of the future outlook of P450BM3 in green chemistry.     

Measurement of saccharification by cellulases

Techniques for screening and measurement of the cellulase complex are reviewed under qualitative or quantitative headings, with emphasis on recent methods of analysis. Appropriate substrates are considered for the constituent enzymes of the complex in the light of physical constraints on hydrolysis.     

Amino acid and peptide derivatives of dimethyl 5-aminoisophthalate as fluorogenic substrates for proteinases

A number of amino acid and peptide derivatives of the fluorophore, dimethyl 5-aminoisophthalate have been synthesized, characterized and tested as substrates for the plant cysteine proteinases papain, ficin and bromelain. In every case, replacement of alanine by citrulline, in the position adjacent to the dimethyl 5-aminoisophthalate resulted in a higher rate of hydrolysis. The partly deprotected dipeptide derivative dimethyl phenylalanylcitrulline-5-aminoisophthalate was hydrolysed most rapidly of all the compounds tested, and on this basis may provide a useful substrate for the detection and quantitative assay of these enzymes.     

Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.     

Degradation of 3-chlorobenzoate by benzoate or 3-methylbenzoate-utilising cultures

Abstract 3-Chlorobenzoate (3CB) was incompletely degraded by bacterial cultures growing continuously with benzoate (Ben) or 3-methylbenzoate (3MB). Accumulation of chlorocatechols as dead-end metabolites was avoided if, prior to the exposure to 3CB, the population had been supplemented with Pseudomonas sp. strain B13 as a chlorocatechol-assimilating member. After acclimatisation, the substrate mixture Ben/3CB was completely degraded via 2 compatible ortho -cleavage pathways.
In contrast, 3MB and 3CB were found to be incompatible substrates: as a result of suicide and genetic inactivation of catechol 2,3-dioxygenase, methylcatechols are subject to unproductive ortho -cleavage. In a defined mixed culture ( Pseudomonas putida mt-2 plus strain B13), 4-carboxymethyl-2-methylbut-2-en-4-olide and 4-carboxymethyl-4-methylbut-2-en-4-olide were excreted as dead-end products, whereas in an undefined mixed culture, degraders of these metabolites became stable members of the community.
Characteristically, with increasing 3CB load, the relative number of 3CB-degrading organisms increased which were Ben⁺ or 3MB⁺ and which had acquired from Pseudomonas sp. strain B13 the ability to assimilate chlorocatechols.     

Neuroprotective Potential and Chemical Profile of Alternatively Cultivated Ganoderma lucidum Basidiocarps

Various neurodegenerative diseases are the main challenges to the modern medicine and there is a great need for novel, natural, neuroprotective agents. Ganoderma lucidum is a well‐known medicinal mushroom, which health benefits have been confirmed by numerous studies. As demand for its basidiocarps is increased and traditional cultivation on hardwoods is not environmentally friendly and economically justified, finding of alternative substrates is necessary. The aim of the study was to assess the effect of alternative cultivation substrates on the chemical profile of G. lucidum basidiocarps and their capacity to inhibit acetylcholinesterase and tyrosinase, which higher activity is directly associated with neurodegenerative processes. Extracts of basidiocarps cultivated on alternative substrates, especially on clear wheat straw, showed significantly higher inhibition capacities than extracts of commercially‐grown ones. These extracts were considerably different chemically from commercial basidiocarps extracts and even nine new compounds were isolated from them. Our results suggest that cultivation substrate greatly affect the chemical profile and neuroprotective capacity of obtained basidiocarps and wheat straw is a promising cultivation substrate.     

Novel Endogenous Protein Kinase C Substrate Proteins, Neutrinins, in Brain Synaptic Membranes of Maturing Rat

Abstract: A new family of membrane phosphoproteins designated as P9, P12, P15, P16, and P20 with corresponding apparent molecular weights of 9K, 12K, 15K, 16K, and 20K was characterized from rat brain by using in vitro exogenous or endogenous phosphorylation and autoradiography. As the phosphorylation was selectively inhibited by the protein kinase C (PKC) inhibitor PKC_19–31 or Ca²⁺-chelating reagents and again stimulated by the PKC activator phorbol 12,13-dibutyrate, these proteins are thought to be the natural PKC substrates. Because P12, P15, P16, and P20 were neutral proteins (pl 7.0) and specifically distributed in neuronal membranes, the new family of membrane-associated PKC substrate proteins was referred to as neutrinins. Neutrinins were widely distributed in rat brain, being especially plentiful in the spinal cord, medulla oblongata, cerebellum, and midbrain, relatively scanty in the cerebral cortex, but lacking in cytosol of brain areas and cell membrane preparations of peripheral tissues. The expression of the developmental changes of neutrinins has been monitored by the in vitro exogenous phosphorylation approach, i.e., adding purified PKC to a deactivated synaptosomal plasma membrane system. Levels of all the neutrinin proteins in rat cerebral cortex, as represented by P12, P15, and P16, showed an ontogenetic increase from the early postnatal days to the adult. This appears to be correlated with the commencement of synaptogenesis.     

Growth and growth substrate levels in spinach under non-steady state conditions of nitrogen nutrition and light

Spinach plants ( Spinacia oleracea L. cv. Subito) were grown in a complete nutrient solution under ample light intensity (14 h day⁻¹ at 660 μmol m⁻² s⁻¹) before being transferred either to a minus-N solution (experiment 1), or to limiting light conditions (6 h day⁻¹ at 220 μmol m⁻² s⁻¹; experiment 2). Shoot growth in experiment 1 decreased significantly from 0.24 day⁻¹ to 0.07 day⁻¹ after the fourth day of transfer. Root relative growth rate increased after 1 day from 0.25 to 0.31 day⁻¹, but decreased on the fifth day after transfer to 0.11 day⁻¹. Shoot growth in experiment 2 decreased significantly from 0.25 to 0.17 day⁻¹ after the fourth day of transfer, while root growth decreased to half of its original level (0.25 day⁻¹) already on the second day. Growth substrate levels in the plants (free sugars, free amino acids) and starch levels depended on the plant age, the moment in the diurnal cycle, and the imposed treatment. Fluctuations in shoot growth or root growth resulting from the light or N limitation could not be explained by a correspondent increase or decrease in the levels of growth substrates. The hypotheses underlying the functional equilibrium theory, assuming shoot and root growth to be controlled by N- and C-containing substrates respectively, and several other growth and partitioning models are therefore questioned. A neglect of the osmotic role of the free sugars in these models might be the explanation for this.