Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes.

Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-ß-1,4-glucanase and ß-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase, licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan, respectively, to component sugars. The pH optima of ß-1,4-glucosidase, endo-ß-1,4-glucanase and total cellulase from G. natalis were 4–5.5, 5.5 and 5.5–7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4–7, 5.5–7 and 4–9, respectively. The pH of the digestive juice was 6.69±0.03 for G. natalis and 6.03±0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-ß-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. ß-1,4-glucosidase from G. natalis was inhibited less by glucono-D-lactone (Ki=11.12 mmol l-1) than was the ß-1,4-glucosidase from D. hirtipes (Ki=4.53 mmol l-1). The greater resistance to inhibition by the ß-1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity of ß-1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

玉米原生质体培养、超低温保存和组织培养的研究

本文叙述了两个玉米基因型（小八趟×水白和白17）原生质体培养的植株再生;用基因型（小八趟×水白）为材料研究影响玉米原生质体培养的各种因素，并此基因型的原生质体经超低温保存后获得植株再生;以及用多种基因型玉米幼胚为材料诱导愈伤组织与植株再生。 影响玉米原生质体游离、分裂与植株再生的因素是多方面的。酶液组合0.2% Onozuka RS + 1% Hemicellulase + 0.1% Pectolyase，利用继代8-16天的愈伤组织，所获原生质体的数量与质量最佳。在原生质体植板率方面，结果表明：N6作为基本培养基是理想的;氮源中，NO3-具有明显的促进作用，而NH4+具有明显的抑制作用;有机氮源是不能缺少的，所使用的四种有机氮源中L-脯氨酸效果最明显。2,4-D浓度以1.0 mg/l最佳。原生质体培养后的渗透压浓度降低的时间以培养四星期后为宜。利用三步诱导，成功地获得胚胎发生的植株再生，并且还指出原生质体起始材料的保存年限大大影响原生质体所再生愈伤组织的分化。 采用上述筛选出的最佳游离、培养以及植株再生的方法，成功地培养了基因型（白17）的原生质体，并获得植株再生。原生质体再生细胞培养4-5天后开始一次分裂;培养15天后，植板率为3-4%。一个月后，原生质体所再生的肉眼可见的愈伤组织，分步转至分化培养基。最后，愈伤组织通过胚胎发生获得植株再生，频率约10%。 玉米原生质体，利用5%DMSO与0.55 M葡萄糖作为混合保护剂，经慢速（1 ℃/分钟）降至－40 ℃，停留二小时后直接投入液氮保存。保存3天后，原生质体在40 ℃的温水浴中快速化冻，成活率高达30-40%。成活原生质体培养后生长正常，植板率高达8-10%。培养5-6星期后，再生愈伤组织转至分化培养基;最后获得植株再生，频率为5-10%。 本文最后叙述了玉米七种基因型的幼胚诱导获得愈伤组织，再生植株频率可达70-80%。 上述各方面的研究结果，对玉米的遗传操作和细胞抗寒性研究、生理代谢的研究等都是十分有价值的。

“Studies on extracellular enzyme production during growth of Pleurotus sp. on lignocellulosic agriwaste and the utilization of spent mushroom substrate”

Commercially, Pleurotus spp. of mushroom are cultivated in bags. After mushroom cultivation, spent substrate remains as residual material. Proper recycling of spent substrate is beneficial for our economy. Spent substrate can be utilized for various other value added purposes through the proper knowledge of its components. Composition of various components depends on the activity of extracellular enzymes in the spent substrate. The present study was conducted to know the enzyme profile of some major extracellular enzymes - cellulase, hemicellulase (xylanase), pectinase and ligninase (lignin peroxidase and laccase) and to estimate cellulose, hemicellulose, pectin and lignin in the substrate. The use of spent substrate as a source of fibre and ethanol, and in the biodegradation of phenol by Pleurotus spp. was also investigated

Effect of a cellulase treatment on extraction of antioxidant phenols from black currant (Ribes nigrum L.) pomace

The effect of a commercial cellulase preparation on phenol liberation and extraction from black currant pomace was studied. The enzyme used, which was from Trichoderma spp., was an effective "cellulase-hemicellulase" blend with low P-glucosidase activity and various side activities. Enzyme treatment significantly increased plant cell wall polysaccharide degradation as well as increasing the availability of phenols for subsequent methanolic extraction. The release of anthocyanins and other phenols was dependent on reaction parameters, including enzyme dosage, temperature, and time. At 50 degrees C, anthocyanin yields following extraction increased by 44% after 3 h and by 60% after 1.5 h for the lower and higher enzyme/substrate ratio (E/S), respectively. Phenolic acids were more easily released in the hydrolytic mixture (supernatant) and, although a short hydrolysis time was adequate to release hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCA) required longer times. The highest E/S value of 0.16 gave a significant increase of flavonol yields in all samples. The antioxidant capacity of extracts, assessed by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, the oxygen radical absorbance capacity, and the ferric reducing antioxidant potential depended on the concentration and composition of the phenols present.

Dietary assimilation and the digestive strategy of the omnivorous anomuran land crab Birgus latro (Coenobitidae).

On Christmas Island, Indian Ocean, the diet of robber crabs, Birgus latro (Linnaeus) was generally high in fat, storage polysaccharides or protein and largely comprised fruits, seeds, nuts and animal material. The plant items also contained significant amounts of hemicellulose and cellulose. In laboratory feeding trials, crabs had similar intakes of dry matter when fed artificial diets high in either fat or storage polysaccharide, but intake was lower on a high protein diet. Assimilation coefficients of dry matter (69–74%), carbon (72–81%), nitrogen (76–100%), lipid (71–96%) and storage polysaccharide (89–99%) were high on all three diets. B. latro also assimilated significant amounts of the chitin ingested in the high protein diet ( 93%) and hemicellulose (49.6–65%) and cellulose (16–53%) from the high carbohydrate and high fat diets. This is consistent with the presence of chitinase, hemicellulase and cellulase enzymes in the digestive tract of B. latro. The mean retention time (27.2 h) for a dietary particle marker (⁵⁷Co-labelled microspheres) was longer than measured in leaf-eating land crabs. The feeding strategy of B. latro involves the selection of highly digestible and nutrient-rich plant and animal material and retention of the digesta for a period long enough to allow extensive exploitation of storage carbohydrates, lipids, protein and significant amounts of structural carbohydrates (hemicellulose, cellulose and chitin).

Anti-amnesic effect of enzyme extracted stevioside from Stevia rebaudiana

The increasing consumption of sucrose has resulted in several nutritional and medicinal problems, including obesity. There is an alarming rise in the prevalence of obesity, type 2 diabetes mellitus, and metabolic syndrome in children and adults around the world, partly related to increasing availability of energy-dense, high-calorie foods, and perhaps to increased consumption of sugar and particularly fructose sweetened beverages. Therefore, low calorie sweeteners are urgently required to substitute table sugar.

Stevioside, a diterpene glycoside, is well known for its intense sweetness and is used as a non-caloric sweetener. Its potential widespread use requires an easy and effective extraction method. Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase, pectinase and hemicellulase using various parameters such as concentration of enzyme, incubation time and temperature was optimized. The extraction conditions were further optimized using response surface methodology (RSM). Under the optimized conditions, the experimental values were in close agreement with predicted model and resulted in a three times yield enhancement of stevioside.

Various studies have revealed that in addition to sweetening nature of stevisoide, it exerts beneficial effects including antihypertensive, anti-hyperglycemic, anti-human rotavirus, antioxidant, anti-inflammatory and antitumor actions. Its anti-amnesic potential remains to be explored, therefore the present study has been undertaken to investigate the beneficial effect of stevioside in memory deficit of rats employing scopolamine induced amnesia as an animal model.

Significance: Stevia is gaining significance in different parts of the world and is expected to develop into a major source of high potency sweetener for the growing natural food market. There is a strong possibility that Stevia sweeteners could replace aspartame in some diet variants. In addition, Stevia is expected to be used as a part substitute for sugar and also used in combination with other artificial sweeteners in the emerging phase of life cycle.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098bp for G. natalis and 1095bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyrisplatycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Production of cellulolytic and hemicellulolytic enzymes from Aureobasidium pulluans on solid state fermentation

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme, remaining 100% active when incubated at 75°C for 1 h. © 2007 Humana Press Inc.

Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

Enzimi, acidi organici ed altri metaboliti coinvolti nella patogenesi di Penicillium spp

Blue mould caused by Penicillium expansum Link is one of the most destructive rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991; Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding, 1972), but their intense use produced the appearance of resistant strains with a great reduction of their activity The aims of the present study were to characterize the isolates of Pencillium sp causing blue mold on pear in Italy by physiological and biochemical parameters. In particular differencing also the behavior of isolates to relationship with sensitivity or resistance to TBZ treatments. We have examined the early stage of infection in relation to enzyme activity, local modulation of pH, production of organic acids, and to secondary metabolism of pathogen. The results described here confirm that the majority of P. expansum isolates from pears packing houses are resistant to TBZ, Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed higher percentage of conidial germination on TBZ-amended medium compared to non amended medium. This may indicate a stimulatory effect of TBZ on conidial germination. Therefore TBZ treatments are not only ineffective for controlling P. expansum, but they may also increase the severity of blue mould on fruits. In the absence of fungicide, isolates showed a significant difference for infection severity, R and RR isolates are characterized by higher pathogenic fitness on fruits, producing larger lesions than S isolates. These data are supported by the study with laboratory-induced resistant isolates, which shows the lack of correlation between TBZ resistance and osmotic sensitivity, and highlights the association between TBZ resistance and infection severity (Baraldi et al 2003). Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity, in addition, the pathogen is able to synthesize a complex enzyme act to degrade the main components of the cell wall especially pectin and cellulose. Isolated sensitive and resistant are characterized by a good activity of pectinase, especially from poligactoronase, which, as already reported by several studies (D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of cell wall. Also, although the measure was minor also highlighted some activities of cellulase, but even note in the production of this kind of cellulase and hemicellulase P. Expansum were not targeted, studies have found no other source of information in this regard. Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid production ability and pH drop. We have found that modulation of pH and the organic acids extrusion were influence to various parameter:  Initial pH: in general, the greatest reduction of pH was observed in isolates grown at pH 7, except for four isolates that maintained the pH of the medium close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1.. In extreme acid condition (pH 3,0) growth and modulation of pH is most lower respect optimal condition (pH 5,0). Also isolates R and RR have showed a greater adaptation to environmental condition more than isolates S.  Time: although the acidification continues for some days, PH modulation is strongest in early hours (48-72 hours)of inoculation process. Time also affects the quality of organic acids, for example in vitro results showed an initial abundant production of succinc acid, followed to important production of galacturoinc acid.  Substrates: there are many differences for the type of acids produced in vitro and in vivo. Results showed in vivo an abundant production of galacturonic, malic, and citric acids and some unknown organic acids in smaller concentrations. Secondary metabolite analysis revealed intra-specific differences, and patulin was found in all isolates, but most significant reduction was observed between in vitro and in vivo samples. There was no correlation between the concentration of patulin, and the percentage of infected fruits, but sample with a lower infection severity of rotten area than the others, showed a significantly lower mycotoxin concentration than samples with a higher lesion diameter of rotten area. Beyond of patulin was detected the presence of another secondary metabolite, penitrem A.

Proceedings of the Eighth Annual Biochemical Engineering Symposium

Contents"Effects of Swell-Shear Treatment on the Digestibility of Cellulosics", Dou-Houng Hwang, UMC "Application of Material and Energy Balance Regularities to Biomass Production from Cellulosic Substrates", Y.H. Lee, KSU "Immobilization of Aspergillus niger beta-Xylosidase", Gbekeloluwa B. Oguntimein, ISU "The Effect of the Major Structural Parameters of Cellulose on Enzymatic Hydrolysis", David H. Beardmore, Y.H. Lee, and L.T. Fan, KSU "Purification of a High Molecular Weight Hemicellulase", Ricardo A. Fournier, ISU "Aerobic Fermentation of Banana Pulp by Aspergillus Fumigatus", Stephen Lorbert, UMC "Purification and Properties of Two Very Small Xylanases", Chih-hen Kiang, ISU "Testing Theoretical Models for Cellulose Enzymatic Hydrolysis", Lin-Chang Chiang, UMC "Utilization of Material and Energy Balances in Hydrocarbon Fermentation", Alexis Ferrer, KSU "Purification of a Series of Closely Related Xylanases", Mary M. Frederick, ISU