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Two important hypotheses concerning the mechanism by which temporal restriction is imposed on the synthesis of enzymes during the cell cycle both suppose that enzyme synthesis occurs during a period of concurrent gene transcription. Halvorson's group have made the remarkable observation that the sequence of enzyme synthesis depends on the sequence of genes on the chromosome1, suggesting that enzyme synthesis depends on a wave of transcription passing along each chromosome. According to the other hypothesis—developed originally for prokaryotic cells2,3—transcription is subject to oscillations in repression (for reviews see Mitchison4 and Halvorson et al.5). A quite different interpretation has also been put forward, that enzyme synthesis is determined by controls acting at the post-transcriptional level6,7.

In Fig. 75 in the 6th lecture we advanced the concept that Pfr in one and the same tissue (and possibly cell) will rapidly induce enzyme synthesis as well as repress enzyme synthesis. We also had to postulate further enzymes whose synthesis or decay are not affected by Pfr.

A suitable simple model tested by experiments is required to address complex biological reaction like esterase synthesis by Saccharomyces cerevisiae. Such an approach might be the answer to a proper bioprocessing strategy. In this regard, a logistic model for esterase production from Saccharomyces cerevisiae has been developed, which predicts well the cell mass, the carbon source (glucose) consumption, and the esterase activity. The accuracy of the model has been statistically examined by using the Student's t-test. The parameter sensitivity analysis showed that all five parameters (?m, Ks, Xm, Yx/s, and Yp/x) have significant influence on the predicted values of esterase activity. � The Korean Society for Microbiology and Biotechnology.

Production of microbial chitinases have received increased attention in recent years due to its potential application in various fields. There has been an all around effort to increase the production of chitinases by using genetically engineered organisms and by incorporating modern fermentation techniques. This review presents a brief outline on the production of microbial chitinases which includes information on random screening and selection of chitinolytic organisms and the rationale behind the use of genetically engineered organisms. The problems and perspectives involved in large scale production of chitinases is discussed with special reference to bioreactor studies and new fermentation methods. The present limitations in the understanding of the chitinase fermentation process is also discussed and the scope for future investigation is outlined in this communication.

The plant pathogenic filamentous hemiascomycete fungus Eremothecium ashbyii is a natural overproducer of riboflavin (vitamin B2). Preceding riboflavin overproduction, microdroplets of lipid were observed in the hyphae of E.ashbyii as yellow fluorescent bodies on staining with the lipid specific dye Nile blue. Following this the fungus wasgrown on different substrates-olive oil, sunflower oil and glucose. Lipid accumulation was followed as a time course by gravimetry. The mycelial lipid was fractionated into triglycerides and phospholipids and relative proportions of constituent fatty acids in them was estimated by GC MS during growth and riboflavin overproduction on each substrate. Changes in mycelial morphology were followed as a time course. In parallel, mycelial growth on each medium was converted to protoplasts whose membrane fluidity was monitored by measuring the fluorescence anisotropy using 1,6 - diphenyl - 1,3,5 - hexatriene (DPH). Lipid accumulation and extracellular lipase activity was maximum at 48 h of growth on all growth substrates. Maximum lipase production coincided with maximum lipid accumulation preceding riboflavin overproduction. A GC-MS analysis of fatty acids during growth (48 h) and riboflavin overproduction (96 h) showed the presence of a large percentage of unsaturated fatty acids in triglycerides and phospholipids. Decrease in Octadecadienoic acid (C 18:2) in the triglycerides during the production phase correlated with riboflavin production while mycelial stability correlated to the Octadecenoic acid (C 18:1) content of the phospholipids. Olive oil-grown mycelia showed maximum lipid accumulation, riboflavin production, lipase activity, membrane fluidity, stability and least morphological changes. Maximum riboflavin was obtained on olive oil medium due to a greater decrease in Octadecadienoic acid content in the triglycerides and the stability of olive oil grown mycelia was attributed to the high content of Octadecenoic acid in its phospholipids.

Extracellular pectin lyase (EC 4.2.2.10) was produced by Xanthomonas campestris in solid state fermentation. Solid state fermentation was carried out using 250 mL Erlenmeyer flasks containing 5 g wheat bran, 1% citrus pectin and 10 mL salt mixture that composed of 0.14% (NH4)2SO4 0.02% MgCl2 and 0.02% K2HPO4. Pectin lyase enzyme was purified 43 fold by using DEAE-cellulose anion exchange column chromatography and characterized. Molecular weight of the enzyme was determined as 77.5 kDa by using Sephadex G-100 gel filtration chromatography. Purification of enzyme was controlled by SDS-PAGE. The optimum pH and temperature of enzyme was determined as pH 9.0 and 50°C, respectively. Pectin lyase was mostly stable 40°C for 24 hours. KM and Vmax were calculated respectively as 0.69 mg/mL and 3.84 |?mol/L?min. Purified pectin lyase was inhibited by Fe+, Zn2+, Ca2+, Co2+ and Hg2+ except for Cu2+ and Mg2+ The purified pectin lyase enzyme was used for getting fruits juices. It was determined that yields of fruits juices improved when it was compared with control.

Rotating simplex method of optimization has been successfully used to determine the optimal conditions for extracellular pectinase production by Aspergillus niger in batch bioreactor. Maximum production of 1.132 U of polymethylgalacturonase, 1.313 U of polygalacturonase and 0.0091 U of pectinlyase was obtained at pH (controlled). 3.38; agitation rate, 483 rpm and aeration rate, 2.12 vvm. These conditions were found to be more suitable for pectinase production.

Tumor embolism occurs in 30 to 50% of all cases of cardiac myxoma, but the causes are still uncertain. Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix (ECM) and play a crucial role in plaque instability and aortic aneurysm development, in addition to cancer and heart failure. To determine whether MMP activity contributes to tumor embolism, we examined 27 left atrium-sided myxomas, 10 of which showed clinical signs of peripheral embolism. Immunohistochemistry (in all cases) and Western blotting, and in situ and in-gel zymography (in four embolic and six nonembolic consecutive tumors) demonstrated higher expression and activity of MT1-MMP, pro-MMP-2, and pro-MMP-9 in embolic myxomas, whereas pro-MMP-1, MMP-3, and TIMP-1 levels were similar to those of nonembolic tumors. Reverse transcriptase-polymerase chain reaction demonstrated that increased MMP activity was due, at least in part, to increased transcription and that TIMP-2 transcripts increased in embolic myxomas. In vitro, embolic tumor cells retained higher MT1-MMP and pro-MMP-2 levels in basal conditions and after stimulation with interleukin-1beta and interleukin-6. Increased MMP synthesis and release correlated with enhanced ECM degradation products containing glycosaminoglycan chains in embolic myxoma tissue. Our results strongly suggest that MMP overexpression may contribute to an excessive degradation of tumor ECM and increase the risk of embolism in cardiac myxomas.

To decrease irreversible thermoinactivation of Aspergillus awamori glucoamylase, five Gly residues causing helix flexibility were replaced with Ala residues. Mutation of Gly57 did not affect thermostability. Mutation of Gly137 doubled it at pHs 3.5 and 4.5 but barely changed it at pH 5.5. The Gly139?Ala mutation did not change thermostability at pH 3.5, improved it at pH 4.5 and worsened it at pH 5.5. The Gly137/Gly139?Ala/Ala mutation gave 1.5-2-fold increased thermostabilities at pus 3.5-5.5. Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137?Ala and Gly137/Gly139?Ala/Ala glucoamylases are the most stable yet produced by mutation. Guanidine treatment at pH 4.5 decreased the reversible stabilities of Gly137?Ala, Gly139?Ala and Gly137/Gly139?Ala/A1a glucoamylases at infinite dilution while not changing those of Gly251?Ala and Gly383?Ala glucoamylases, which is, in general, opposite to what occurred with thermoinactivation. Mutation of Gly57 greatly improved the extracellular glucoamylase production by yeast, that of Gly137 barely affected it and those of Gly139 and of both Gly137 and Gly139 strongly impeded it. These observations suggest that �-helix rigidity can affect reversible and irreversible glucoamylase stability differently, that the effects of multiple mutations within one �-helix to improve stability are not always additive and that even single mutations can strongly affect extracellular enzyme production.

During germination ofSorghum vulgare seeds, inhibitors of pea diamine oxidase appeared in the embryos. One was heat labile, dialysable and inhibited the enzyme in vitro, while another was heat stable and inhibited the enzyme synthesis when pea seeds were soaked and allowed to germinate in the extract containing the inhibitors.

Dexamethasone and prednisone in physiologic range increased angiotensin converting enzyme 7- to 16-fold in comparison to control in 3 days at maximal stimulation (4 nM steroid) in rabbit alveolar macrophages in culture. The increase was inhibited by actinomycin D (0.1 microng/ml) and 1 micronM cycloheximide, suggesting that de novo transcription and enzyme synthesis are responsible for the increased enzyme activity. This result is evidence for a regulatory mechanism for angiotensin converting enzyme, which is important in blood pressure control.

DURING the investigation of metabolism of storage tissues of higher plants, it is common to wash and aerate disks of the tissue for periods of several days or weeks. To this end, a variety of techniques has been reported, but in most cases it was not possible to impose rigorous conditions of sterility, although, in analysing the behaviour of tissues treated in this way, it is evidently necessary to ensure that the presence of micro-organisms is not a significant factor. The experimenter has usually used such criteria as retention of pigment if any, maintenance of turgor, and clarity of the suspending liquid. Recently, Bacon, MacDonald and Knight1 have shown that these criteria may be observed even when substantial contamination of disks by bacteria has occurred, and they found that disks (1 cm diam. and 1 mm thick) of sugar beet showed the presence of about 3 × 107 organisms/disk after washing for 4 days. During a programme of research into enzyme synthesis in storage tissue disks2,3 we have found that artichoke and carrot disks become contaminated with bacteria to a similar degree, ranging from 5 × 105 per disk after 24 h to 4 × 107 after 3 days.

Treatment of hamster embryonic cells with 1,2-benzanthracene for 4 to 48 hours induced a three- to tenfold increase in the activity of benzpyrene hydroxylase. That the increase in enzyme activity was completely prevented by puromycin suggested an induction of enzyme synthesis.

Alternative theories for the synthesis of enzyme during the cell cycle lead to two regression models for the increase in enzyme concentration in synchronous cultures of yeast cells. One model is a segmented linear regression with three segments; the alternative is a smooth exponential. The analysis of observations from a series of experiments designed to discriminate between the alternative models is discussed. Methods are described for estimating the segmented linear regression and for determining by simulation the distributions under the two models of the discriminating criterion, the ratio of maximised likelihoods. The consequences of non-validity of some of the assumptions made in setting up the regression models are investigated.

The lag phase between application of gibberellic acid to barley aleurone layers and the beginning of amylase synthesis is at a minimum at 30 °C. This temperature is also the optimum for enzyme synthesis. Incubation temperature has little effect on the background activity found in the absence of gibberellic acid, or on the concentration of enzyme retained in the tissue in the presence of hormone. When aleurone layers spend only 2 h of the lag period at 30 °C, and the remainder of the incubation at 25 or 15 °C, the lag period is considerably reduced. Short-term incubation at 30 °C is most effective in reducing the lag period at 25 °C when given for the first 2 h after gibberellic-acid addition. Under these conditions ('tempera ture stepdown incubation') the lag period is reduced to that with the whole incubation at 30 °C. The temperature stepdown incubation has a sharp pH optimum of 5-00-5-05 and its effectiveness is increased by the presence of iron in the medium. There are thus two rate limiting reactions during the lag period, the first highly temperature sensitive, the second relatively temperature insensitive.