Critical Reviews in Biotechnology

Published by Informa Healthcare
Online ISSN: 1549-7801
Print ISSN: 0738-8551
Publications
Human gene therapy and its application for the treatment of human genetic disorders, such as cystic fibrosis, cancer, and other diseases, are discussed. Gene therapy is a technique in which a functioning gene is inserted into a human cell to correct a genetic error or to introduce a new function to the cell. Many methods, including retroviral vectors and non-viral vectors, have been developed for both ex vivo and in vivo gene transfer into cells. Vectors need to be developed that efficiently transfer genes to target cells, and promoter systems are required that regulate gene expression according to physiologic needs of the host cell. There are several safety and ethical issues related to manipulating the human genome that need to be resolved. Current gene therapy efforts focus on gene insertion into somatic cells only. Gene therapy has potential for the effective treatment of genetic disorders, and gene transfer techniques are being used for basic research, for example, in cancer, to examine the underlying mechanism of disease. There are still many technical obstacles to be overcome before human gene therapy can become a routine procedure. The current human genome project provides the sequences of a vast number of human genes, leading to the identification, characterization, and understanding of genes that are responsible for many human diseases.
 
Selenium (Se) is an essential trace element unevenly distributed on the Earth's crust with low selenium regions predominating. To prevent selenium-deficiency diseases in livestock, additions of selenium to animal feed are required and were approved for all species, but the chemical form of the element to be added was not specified. Presently, sodium selenite is still widely employed, although it is not a natural nutritional form of selenium. Its use creates ecological problems and affects human selenium nutriture in as much as the meat, milk, and eggs from animals maintained on selenite contain less selenium than from animals receiving it as selenomethionine, the chief natural nutritional form of the element present in grain crops grown in selenium-adequate regions, or from high-selenium yeast added to feedstock. Human dietary selenium intakes are sub-optimal in many countries but are considered to be adequate if they reach the currently adopted Recommended Dietary Allowances (RDAs). Their upward revision will be required if the health benefits of selenium are to be fully utilized.
 
Nanotechnology is emerging as a field of applied science and technology. Synthesis of nanoparticles is done by various physical and chemical methods but the biological system is gaining attention as an eco-friendly technique. The biosynthetic method employing plant parts is proving as a simple and cost-effective method for the synthesis of nanoparticles. The present mini review focuses on the different systems utilized for the synthesis of nanoparticles with special emphasis on the use of plants for the synthesis process, its applications and future directions.
 
Abstract Cyanobacteria have developed various response mechanisms in long evolution to sense and adapt to external or internal changes under abiotic stresses. The signal transduction system of a model cyanobacterium Synechocystis sp. PCC 6803 includes mainly two-component signal transduction systems of eukaryotic-type serine/threonine kinases (STKs), on which most have been investigated at present. These two-component systems play a major role in regulating cell activities in cyanobacteria. More and more co-regulation and crosstalk regulations among signal transduction systems had been discovered due to increasing experimental data, and they are of great importance in corresponding to abiotic stresses. However, mechanisms of their functions remain unknown. Nevertheless, the two signal transduction systems function as an integral network for adaption in different abiotic stresses. This review summarizes available knowledge on the signal transduction network in Synechocystis sp. PCC 6803 and biotechnological implications under various stresses, with focuses on the co-regulation and crosstalk regulations among various stress-responding signal transduction systems.
 
In the past few years, the signal transduction of the plant hormone abscisic acid (ABA) has been studied extensively and has revealed an unanticipated complex. ABA, characterized as an intracellular messenger, has been proven to act a critical function at the heart of a signaling network operation. It has been found that ABA plays an important role in improving plant tolerance to cold, as well as triggering leaf senescence for years. In addition, there have been many reports suggesting that the signaling pathways for leaf senescence and plant defense responses may overlap. Therefore, the objective was to review what is known about the involvement of ABA signaling in plant responses to cold stress and regulation of leaf senescence. An overview about how ABA is integrated into sugars and reactive oxygen species signaling pathways, to regulate plant cold tolerance and leaf senescence, is provided. These roles can provide important implications for biotechnologically improving plant cold tolerance.
 
Among different liquid biofuels that have emerged in the recent past, biobutanol produced via fermentation processes is of special interest due to very similar properties to that of gasoline. For an effective design, scale-up, and optimization of the acetone-butanol-ethanol (ABE) fermentation process, it is necessary to have insight into the micro- and macro-mechanisms of the process. The mathematical models for ABE fermentation are efficient tools for this purpose, which have evolved from simple stoichiometric fermentation equations in the 1980s to the recent sophisticated and elaborate kinetic models based on metabolic pathways. In this article, we have reviewed the literature published in the area of mathematical modeling of the ABE fermentation. We have tried to present an analysis of these models in terms of their potency in describing the overall physiology of the process, design features, mode of operation along with comparison and validation with experimental results. In addition, we have also highlighted important facets of these models such as metabolic pathways, basic kinetics of different metabolites, biomass growth, inhibition modeling and other additional features such as cell retention and immobilized cultures. Our review also covers the mathematical modeling of the downstream processing of ABE fermentation, i.e. recovery and purification of solvents through flash distillation, liquid-liquid extraction, and pervaporation. We believe that this review will be a useful source of information and analysis on mathematical models for ABE fermentation for both the appropriate scientific and engineering communities.
 
Abstract The beneficial effects of endophytes on plant growth are important for agricultural ecosystems because they reduce the need for fertilizers and decrease soil and water pollution while compensating for environmental perturbations. Endophytic fungi are a novel source of bioactive secondary metabolites; moreover, recently they have been found to produce physiologically active gibberellins as well. The symbiosis of gibberellins producing endophytic fungi with crops can be a promising strategy to overcome the adverse effects of abiotic stresses. The association of such endophytes has not only increased plant biomass but also ameliorated plant-growth during extreme environmental conditions. Endophytic fungi represent a trove of unexplored biodiversity and a frequently overlooked component of crop ecology. The present review describes the role of gibberellins producing endophytic fungi, suggests putative mechanisms involved in plant endophyte stress interactions and discusses future prospects in this field.
 
Multiple functions of aquaporins in relation to different plant parts. The panel-A describes the substrates transported through aquaporins in different plant cells. In root cell, aquaporins mediate the uptake of water, nutrients and toxic heavy metals from soil solution, while in leaf cell their major function is to facilitate water and CO 2 transport. In both these two organs, aqua- 
Abstract Abiotic stress has become a challenge to food security due to occurrences of climate change and environmental degradation. Plants initiate molecular, cellular and physiological changes to respond and adapt to various types of abiotic stress. Understanding of plant response mechanisms will aid in strategies aimed at improving stress tolerance in crop plants. One of the most common and early symptoms associated with these stresses is the disturbance in plant-water homeostasis, which is regulated by a group of proteins called "aquaporins". Aquaporins constitute a small family of proteins which are classified further on the basis of their localization, such as plasma membrane intrinsic proteins, tonoplast intrinsic proteins, nodulin26-like intrinsic proteins (initially identified in symbiosomes of legumes but also found in the plasma membrane and endoplasmic reticulum), small basic intrinsic proteins localized in ER (endoplasmic reticulum) and X intrinsic proteins present in plasma membrane. Apart from water, aquaporins are also known to transport CO2, H2O2, urea, ammonia, silicic acid, arsenite and wide range of small uncharged solutes. Besides, aquaporins also function to modulate abiotic stress-induced signaling. Such kind of versatile functions has made aquaporins a suitable candidate for development of transgenic plants with increased tolerance toward different abiotic stress. Toward this endeavor, the present review describes the versatile functions of aquaporins in water uptake, nutrient balancing, long-distance signal transfer, nutrient/heavy metal acquisition and seed development. Various functional genomic studies showing the potential of specific aquaporin isoforms for enhancing plant abiotic stress tolerance are summarized and future research directions are given to design stress-tolerant crops.
 
Reactive oxygen species (ROS) are produced in plants as byproducts during many metabolic reactions, such as photosynthesis and respiration. Oxidative stress occurs when there is a serious imbalance between the production of ROS and antioxidant defense. Generation of ROS causes rapid cell damage by triggering a chain reaction. Cells have evolved an elaborate system of enzymatic and nonenzymatic antioxidants which help to scavenge these indigenously generated ROS. Various enzymes involved in ROS-scavenging have been manipulated, over expressed or downregulated to add to the present knowledge and understanding the role of the antioxidant systems. The present article reviews the manipulation of enzymatic and nonenzymatic antioxidants in plants to enhance the environmental stress tolerance and also throws light on ROS and redox signaling, calcium signaling, and ABA signaling.
 
The fast pace of technological change in the biotechnology industry and the market demands require continuous innovation, which, owing to the science base of the sector, derives from academic research through a transformation process that converts science-oriented knowledge to marketable products. There appear to be some inherent difficulties in transforming directly the knowledge output of academic research to industrial use. The purpose of this article is to examine certain transition mechanisms from monodisciplinary academic isolation (curiosity-driven and internal-worth innovation) to university-industry alliances (market-driven and public-worth innovation) through inter-organizational multidisciplinary collaboration and contextualize the analysis with the case of biosensors. While the majority of literature on the subject studies the channels of knowledge transfer as determinants of alliance success (transferor/transferee interactions), either from the university side (science base) or the industry side (market base), this article focuses on the transferable (technology base) and how it can be strategically modeled and managed by the industry to promote innovation. Based on the valuable lessons learnt from the biosensor paradigm, the authors argue that strategic industry choices deal primarily with the best stage/point to intersect and seize the university output, implanting the required element of marketability that will transform an idea to a viable application. The authors present a methodological approach for accelerating the knowledge transfer from the university to industry aiming at the effective transition of science to products through a business model reconfiguration.
 
Different proteolytic enzymes in cheese during ripening (adapted from Sousa et al., 2001).  
Basic cheese ripening biochemistry (adapted from Law, 2001).  
Commercial enzymes to accelerate ripening of cheese
Cheese is one of the dairy products that can result from the enzymatic coagulation of milk. The basic steps of the transformation of milk into cheese are coagulation, draining, and ripening. Ripening is the complex process required for the development of a cheese's flavor, texture and aroma. Proteolysis, lipolysis and glycolysis are the three main biochemical reactions that are responsible for the basic changes during the maturation period. As ripening is a relatively expensive process for the cheese industry, reducing maturation time without destroying the quality of the ripened cheese has economic and technological benefits. Elevated ripening temperatures, addition of enzymes, addition of cheese slurry, attenuated starters, adjunct cultures, genetically engineered starters and recombinant enzymes and microencapsulation of ripening enzymes are traditional and modern methods used to accelerate cheese ripening. In this context, an up to date review of Cheddar cheese ripening is presented.
 
The acetic acid bacteria (AAB) have important roles in food and beverage production, as well as in the bioproduction of industrial chemicals. In recent years, there have been major advances in understanding their taxonomy, molecular biology, and physiology, and in methods for their isolation and identification. AAB are obligate aerobes that oxidize sugars, sugar alcohols, and ethanol with the production of acetic acid as the major end product. This special type of metabolism differentiates them from all other bacteria. Recently, the AAB taxonomy has been strongly rearranged as new techniques using 16S rRNA sequence analysis have been introduced. Currently, the AAB are classified in ten genera in the family Acetobacteriaceae. AAB can not only play a positive role in the production of selected foods and beverages, but they can also spoil other foods and beverages. AAB occur in sugar- and alcohol-enriched environments. The difficulty of cultivation of AAB on semisolid media in the past resulted in poor knowledge of the species present in industrial processes. The first step of acetic acid production is the conversion of ethanol from a carbohydrate carried out by yeasts, and the second step is the oxidation of ethanol to acetic acid carried out by AAB. Vinegar is traditionally the product of acetous fermentation of natural alcoholic substrates. Depending on the substrate, vinegars can be classified as fruit, starch, or spirit substrate vinegars. Although a variety of bacteria can produce acetic acid, mostly members of Acetobacter, Gluconacetobacter, and Gluconobacter are used commercially. Industrial vinegar manufacturing processes fall into three main categories: slow processes, quick processes, and submerged processes. AAB also play an important role in cocoa production, which represents a significant means of income for some countries. Microbial cellulose, produced by AAB, possesses some excellent physical properties and has potential for many applications. Other products of biotransformations by AAB or their enzymes include 2-keto-L-gulonic acid, which is used for the production of vitamin C; D-tagatose, which is used as a bulking agent in food and a noncalorific sweetener; and shikimate, which is a key intermediate for a large number of antibiotics. Recently, for the first time, a pathogenic acetic acid bacterium was described, representing the newest and tenth genus of AAB.
 
Biochemical pathway of light dependant synthesis of fatty acids and synthesis of triacylglycerides, phospholipids, and galactolipids in plants (Enzymes: AAT, acyl-ACP thioesterase; ACCase, acetyl-CoA carboxylase; ACS, acetyl-CoA synthase; DGAT, acylCoA:diacylglycerol acyltransferase; DGD, DGDG synthase; FAS, type II fatty acid synthase; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidate acyltransferase; MGD, MGDG synthase; PAP, phosphatidic acid phosphatase; plPDC, plastid pyruvate dehydrogenase complex; SACPD, stearoyl-ACP desaturase. Substrates and products: DAG, diacylglycerol; TAG, triacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol. (Illustration based on Ohlrogge and Jaworski (1997), Murphy (1999), Awai et al. (2007), Courchesne et al. (2009), Joyard et al. (2010) and Khozin-Goldberg and Cohen (2011).
ACCase taxonomy showing first, second, and third generation plastids in different algal taxa and the current state of knowledge on the presence of heteromeric or homomeric ACCase in plastids (figure modified from Wilson (2005)). 
(A) Algae with first generation plastid, (B) algae with second generation plastid with three membranes, (C) algae with second generation plastid with four membranes, two of which form the chloroplast endoplasmic reticulum, and (D) algae with second generation chloroplast with four membranes, two of which form the chloroplast endoplasmic reticulum (CER) membrane, including a nucleomorph. N2 host nucleus, 1. Plastid envelope derived from primary endosymbiotic event, 2. Bacterial chromosome, 3. Prokaryotic ribosomes, 4. Plastid envelope derived from secondary endosymbiotic event, 5. CER outer membrane continuous with nuclear envelope and inner membrane compartmentalizing the plastid from the ER, 6. Nucleomorph: remnant nucleus N1, of eukaryotic symbiont (present only in Cryptophyta and Chlorarachniphyta), 7. eukaryotic ribosomes.
Carboxylbiotin binding region of β-carboxyl transferase (Roessler and Ohlrogge 1993). Grey highlights the most conservative regions within the heteromeric and homomeric forms. Blue and green species names denote homomeric ACCase positively identified to be found in the cytosol and plastid, respectively. Solid lines identify conserved regions in both heteromeric and homomeric ACCase.
Lipids from microalgae have become an important commodity in the last 20 years, biodiesel and supplementing human diets with ω-3 fatty acids are just two of the many applications. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the lipid synthesis pathway. In general, ACCases consist of four functional domains: the biotin carboxylase (BC), the biotin carboxyl binding protein (BCCP), and α-and ß-carboxyltransferases (α-and ß-CT). In algae, like in plants, lipid synthesis is another function of the chloroplast. Despite being well researched in plants and animals, there is a distinct lack of information about this enzyme in the taxonomically diverse algae. In plastid-containing organisms, ACCases are present in the cytosol and the plastid (chloroplasts) and two different forms exist, the heteromeric (prokaryotic) and homomeric (eukaryotic) form. Despite recognition of the existence of the two ACCase forms, generalized published statements still list the heteromeric form as the one present in algal plastids. In this study, the authors show this is not the case for all algae. The presence of heteromeric or homomeric ACCase is dependent on the origin of plastid. The authors used ACCase amino acid sequence comparisons to show that green (Chlorophyta) and red (Rhodophyta) algae, with the exception of the green algal class Prasinophyceae, contain heteromeric ACCase in their plastids, which are of primary symbiotic origin and surrounded by two envelope membranes. In contrast, algal plastids surrounded by three to four membranes were derived through secondary endosymbiosis (Heterokontophyta and Haptophyta), as well as apicoplast containing Apicomplexa, contain homomeric ACCase in their plastids. Distinctive differences in the substrate binding regions of heteromeric and homomeric α-CT and β-CT were discovered, which can be used to distinguish between the two ACCase types. Furthermore, the acetyl-CoA binding region of homomeric α-CT can be used to distinguish between cytosolic and plastidial ACCase. The information provided here will be of fundamental importance in ACCase expression and activity research to unravel impacts of environmental and physicochemical parameters on lipid content and productivity.
 
The cytoplasmic membranes of many aerobic and facultative bacteria contain enzymes that catalyze the reduction of dissolved oxygen to water. Preparations of small particles derived from such membranes can be filter sterilized without loss of the oxygen-reducing enzymes. These particle preparations can be used to produce anaerobic conditions in a variety of biological environments. They have been shown to stimulate the growth of many anaerobic bacteria and can also be used to stabilize oxygen-sensitive chemical reagents. The particle preparations are stable for long periods of time. They are functional over a pH range and temperature range frequently encountered in biological systems. Various techniques for using the particles are presented. The advantages and limitations of this new approach to achieving oxygen-free conditions are discussed.
 
The production of organic acids covers two aspects: first, the metabolic pathways involved in the biosynthesis, and, second, the industrial process strategy adopted. The review seeks to show the underlying biochemical similarities in the biosynthesis of organic acids and the resulting similarities in the commercial processes. Two groups of acids are defined, those with a "long" biosynthetic path from glucose, involving much of the glycolytic pathway and the tricarboxylic acid cycle, and those acids with a "short pathway", essentially a biotransformation of glucose. The regulation of the pathways and the future developments in metabolic control theory and genetic manipulations relating to them are considered. The organisms used industrially are also limited, Aspergillus sp. and Candida yeasts; again the underlying metabolic similarities lead to similar strategies for all the acids discussed.
 
Properties and significant market data of emerging high-value organic acids.
Comparison of noteworthy recent research studies on the bio-based production of organic acids using different microbial platforms and feedstocks.
Continued
Summary of noteworthy recent advances in developing efficient microbial platforms through metabolic engineering for producing high-value organic acids.
Abstract Microbial production of organic acids has become a fast-moving field due to the increasing role of these compounds as platform chemicals. In recent years, the portfolio of specialty fermentation-derived carboxylic acids has increased considerably, including the production of glyceric, glucaric, succinic, butyric, xylonic, fumaric, malic, itaconic, lactobionic, propionic and adipic acid through innovative fermentation strategies. This review summarizes recent trends in the use of novel microbial platforms as well as renewable and waste materials for efficient and cost-effective bio-based production of emerging high-value organic acids. Advances in the development of robust and efficient microbial bioprocesses for producing carboxylic acids from low-cost feedstocks are also discussed. The industrial market scenario is also reviewed, including the latest information on the stage of development for producing these emerging bio-products via large-scale fermentation.
 
Polyunsaturated fatty acids like EPA and DHA have attracted a great attention due to their beneficial effects on human health. At present, fish oil is the major source of EPA and DHA. Various alternative sources are being explored to get these essential fatty acids. Genes encoding enzymes involved in the biosyntheses of PUFAs have been identified, cloned and gene prospecting becomes a novel method for enhanced PUFA production. Desaturase and elongase genes have important biotechnological appeal from genetic engineering point of view. This review highlights the research and results on such enzymes.
 
More than 40 years ago, it was reported that methionine markedly stimulated production of cephalosporin C by Cephalosporium acremonium. Over the years, many hypotheses were put forth to explain this phenomenon. The accumulating evidence strongly supported the concept that methionine stimulates by inducing enzymes of the biosynthetic pathway such as delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase. This mechanism has been strengthened by the finding that transcription of the genes encoding the above enzymes is markedly enhanced by growth with methionine. An effect of methionine in the fermentation unrelated to the titer stimulation is its contribution of the sulfur atom to the cephalosporin molecule. Methionine also stimulates mycelial fragmentation; the relationship between this effect on hyphal differentiation and the induction of the cephalosporin synthases remains to be elucidated.
 
The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.
 
New antimicrobial agents are desperately needed to combat the increasing number of antibiotic resistant strains of pathogenic microorganisms. Natural products remain the most propitious source of novel antibiotics. It is widely accepted that actinobacteria are prolific producers of natural bioactive compounds. We argue that the likelihood of discovering a new compound having a novel chemical structure can be increased with intensive efforts in isolating and screening rare genera of microorganisms. Screening rare actinomycetes and their previously under-represented genera from unexplored environments in natural product screening collections is one way of achieving this. Rare actinomycetes are usually regarded as the actinomycete strains whose isolation frequency is much lower than that of the streptomycete strains isolated by conventional methods. Many natural environments are still either unexplored or under-explored and thus, can be considered as a prolific resource for the isolation of less exploited microorganisms. More and different ecological niches need to be studied as sources of a greater diversity of novel microorganisms. In this review, we wish to update our understanding of the potential of the rare actinomycetes by focusing on the ways and means of enhancing their bio-discovery potential.
 
Actinomycetes represent the microbial group richest in production of variable secondary metabolites. These mostly bioactive molecules are the end products of complex multistep biosynthetic pathways. Recent progress in the molecular genetics and biochemistry of the biosynthetic capacities of actinomycetes enables first attempts to redesign these pathways in a directed fashion. However, in contrast to several examples of designed biochemical improvement of primary metabolic processes in microorganisms, none of the products or strains derived from pathway engineering in actinomycetes discussed herein have reached pilot or production scale. The main reasons for this slow progress are the complicated pathways themselves, their complex regulation during the actinomycete cell cycle, and their uniqueness, as most pathways and products are specific for a strain rather than for a given species or larger taxonomic group. However, the modular use of a minimum of very similar enzymes and their conversion of similar intermediates to form the building blocks for the production of a maximum of divergent end products gives hope for the future application of these genetic models for the redesign of complex pathways for modified or new natural products. Several strategies that can be followed to reach this aim are discussed, mainly for the variable 6-deoxyhexose metabolism as an ubiquitously applicable example.
 
The Gram-positive bacterial genus Streptomyces possesses interesting biological aspects, such as the ability to produce a wide variety of secondary metabolites and a mycelial form of growth that culminates in sporulation. A close relationship of secondary metabolism and cell differentiation has been well recognized; secondary metabolism might be a physiological expression of cell differentiation. A variety of diffusible low-molecular-weight chemical substances have been found to function in general as regulatory factors, like "hormones" in eukaryotes, for secondary metabolism and cell differentiation. Among these factors, A-factor has been most extensively studied. This review summarizes recent research on the chemical structures, functions, biosyntheses, and mode of action of these regulatory factors.
 
Mechanisms of different killer toxin action. 
A and C: The sensitive cells treated with the purified killer toxins from K. siamensis HN12-1 and M. frigida 2E00797, respectively. B and D: the control sensitive cells.
Abstract Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.
 
The mammalian serine protease zymogen, plasminogen, can be converted into the active enzyme plasmin by vertebrate plasminogen activators urokinase (uPA), tissue plasminogen activator (tPA), factor XII-dependent components, or by bacterial streptokinase. The biochemical properties of the major components of the system, plasminogen/plasmin, plasminogen activators, and inhibitors of the plasminogen activators, are reviewed. The plasmin system has been implicated in a variety of physiological and pathological processes such as fibrinolysis, tissue remodeling, cell migration, inflammation, and tumor invasion and metastasis. A defective plasminogen activator/inhibitor system also has been linked to some thromboembolic complications. Recent studies of the mechanism of fibrinolysis in human plasma suggest that tPA may be the primary initiator and that overall fibrinolytic activity is strongly regulated at the tPA level. A simple model for the initiation and regulation of plasma fibrinolysis based on these studies has been formulated. The plasminogen activators have been used for thrombolytic therapy. Three new thrombolytic agents--tPA, pro-uPA, and acylated streptokinase-plasminogen complex--have been found to possess better properties over their predecessors, urokinase and streptokinase. Further improvements of these molecules using genetic and protein engineering tactics are being pursued.
 
Plasminogen activators (PAs) are proteases that convert plasminogen to plasmin. Plasmin, in turn, is a protease that can lyse a fibrin clot and, therefore, PAs have a primary role in fibrinolysis. Two PAs, urokinase (UK) and streptokinase (SK), have been available for therapeutic use for years. Unfortunately, both can cause systemic fibrinogenolysis and other side effects which have limited their use. Interest has focused on a different enzyme, tissue plasminogen activator (t-PA), which will cause specific clot lysis without systemic problems. The gene for t-PA has been cloned and many biotechnology firms are preparing to produce t-PA for therapeutic use. The properties and potential for therapy of t-PA are reviewed and compared to new forms of other activators, such as pro-urokinase. How the interactions of PAs and inhibitors may affect the use of PAs is also discussed.
 
Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.
 
Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and beta-lactamase are also covered.
 
Structure of (a) processed (Protein Data Bank Code 1PNK) and (b) slow-processing mutant precursor (PDB code 1E3A) of EcPGA. (The slow-processing mutant precursor retains the spacer peptide). The important residues have been highlighted.
Characteristics of penicillin G acylases and related enzymes from different bacteria.
Characteristics of penicillin V acylases and related enzymes.
Reaction mechanism of penicillin acylases (amino acids based on B. sphaericus PVA). The sulfhydryl group of the N-terminal cysteine (stabilized by Arg18) performs a nucleophilic attack (1) on the carbon atom of the amide bond of penicillin and related substrates. The tetrahedral intermediate is stabilized by the oxyanion hole (2), involving Asn175 and Tyr82. After the release of the leaving amino product (3), the second nucleophilic attack is carried out by a protonated water molecule on the thioester bond between Cys2 and the substrate. A similar tetrahedral intermediate is formed (5), eventually leading to the liberation of carboxylic acid and regeneration of free enzyme (6). 
Overlap of the 8 loops surrounding the catalytic site in BlBSH (Bifidobacterium longum) and BsuPVA (Bacillus subtilis). The front view and side view are shown. Four loops 1, 2, 5 and 8 show major differences in length (inset) and play a crucial role in determining the substrate specificity.
Abstract It is of great importance to study the physiological roles of enzymes in nature; however, in some cases, it is not easily apparent. Penicillin acylases are pharmaceutically important enzymes that cleave the acyl side chains of penicillins, thus paving the way for production of newer semi-synthetic antibiotics. They are classified according to the type of penicillin (G or V) that they preferentially hydrolyze. Penicillin acylases are also used in the resolution of racemic mixtures and peptide synthesis. However, it is rather unfortunate that the focus on the use of penicillin acylases for industrial applications has stolen the spotlight from the study of the importance of these enzymes in natural metabolism. The penicillin acylases, so far characterized from different organisms, show differences in their structural nature and substrate spectrum. These enzymes are also closely related to the bacterial signalling phenomenon, quorum sensing, as detailed in this review. This review details studies on biochemical and structural characteristics of recently discovered penicillin acylases. We also attempt to organize the available insights into the possible in vivo role of penicillin acylases and related enzymes and emphasize the need to refocus research efforts in this direction.
 
Some Oligopeptide Epitopes with Clinical Significance 
A hypothetical cooperative mechanism of endogenous cytolysin targeting mediated via some RMM: (a) cell-cell interaction; (b) influx of Ca 2 • and RMM dissociation following a decrease in Ca 2 • concentration; (c) secretion of cytolysins and their interaction with the target coated by RMM and membrane perforation; (d) endocytosis; (e) subsequent events (Kubrycht et al., 1993) (Sections IX and X). 
This review summarizes some important data, principles, opinions, commentaries, and modern methodology concerning the receptor structure, interactions, signaling and receptor-mediated cell functions. Three sections give a brief overview of the signaling synergy, multivariant signaling, and some reactions in phosphorylation networks. A concise report about the cytotoxic reaction of NK cells represents an example of multistage recognition reaction, involving differently acting receptors, changes in affinities of cell-cell interactions, and secretion of regulatory and cytotoxic molecules. Some interesting trends in receptor engineering, including antibody molecules as a special receptor phenomenon are mentioned in the final section.
 
Corynebacterium glutamicum and its close relatives, C. flavum and C. lactofermentum, have been used for over 3 decades in the industrial production of amino acids by fermentation. Since 1984, several research groups have started programs to develop metabolic engineering principles for amino acid-producing Corynebacterium strains. Initially, the programs concentrated on the isolation of genes encoding (deregulated) biosynthetic enzymes and the development of general molecular biology tools such as cloning vectors and DNA transfer methods. With most of the genes and tools now available, recombinant DNA technology can be applied in strain improvement. To accomplish these improvements, it is critical and advantageous to understand the mechanisms of gene expression and regulation as well as the biochemistry and physiology of the species being engineered. This review explores the advances made in the understanding and application of amino acid-producing bacteria in the early 1990s.
 
Sea buckthorn is a berry crop with multiple uses. The berries are highly appreciated for their unique taste but are also very rich in bioactive compounds with powerful nutritional and medicinal values. In addition, the plants grow well under adverse conditions, and are often used to fight soil erosion. Utilization of sea buckthorn has therefore increased around the world but serious problems have, nevertheless, been encountered due to drought, salinity, diseases and insect pests. This review covers important aspects of sea buckthorn research, such as heritable and environmentally induced variation in biochemical compounds, causes and effects of the devastating dried-shrink disease, susceptibility to insect pests, methods for conventional breeding, and the utilization of DNA markers for taxonomical and population genetic analyses, and for investigating the inheritance of quality and resistance traits. We also present possibilities to implement innovative biotechnological breeding methods, especially metabolite profiling and MAS/GRC-based markers, for fast and efficient development of elite genotypes with specific nutritional- and health-related bioactive compounds and strong resistance to biotic and abiotic stress.
 
Metabolic pathways for H 2 production in Chlamydomonas reinhardtii cells. H 2 can be produced by both direct water photolysis operated 
Experiments of H 2 production with the C. reinhardtii D239–40 
Overview of the 50L horizontal tubular photo-bioreactor used for outdoor experiments with C. reinhardtii (CC124). Insert a culture during the H 2 production phase. 
Time course of pH response to the injection of a concentrated HCl solution enters the 50L PBR (a); and 110L PBR (b). The dashed lines indicate the 0.05 DpH (t 0 ) conditions used to calculate the mixing time.
Abstract Biological hydrogen production is being evaluated for use as a fuel, since it is a promising substitute for carbonaceous fuels owing to its high conversion efficiency and high specific energy content. The basic advantages of biological hydrogen production over other "green" energy sources are that it does not compete for agricultural land use, and it does not pollute, as water is the only by-product of the combustion. These characteristics make hydrogen a suitable fuel for the future. Among several biotechnological approaches, photobiological hydrogen production carried out by green microalgae has been intensively investigated in recent years. A select group of photosynthetic organisms has evolved the ability to harness light energy to drive hydrogen gas production from water. Of these, the microalga Chlamydomonas reinhardtii is considered one of the most promising eukaryotic H2 producers. In this model microorganism, light energy, H2O and H2 are linked by two excellent catalysts, the photosystem 2 (PSII) and the [FeFe]-hydrogenase, in a pathway usually referred to as direct biophotolysis. This review summarizes the main advances made over the past decade as an outcome of the discovery of the sulfur-deprivation process. Both the scientific and technical barriers that need to be overcome before H2 photoproduction can be scaled up to an industrial level are examined. Actual and theoretical limits of the efficiency of the process are also discussed. Particular emphasis is placed on algal biohydrogen production outdoors, and guidelines for an optimal photobioreactor design are suggested.
 
Over the past decades a large amount of biopolymers originating from various types of microorganisms have been reported. With ongoing research the number of possible applications has increased rapidly, ranging from use as food additives and biomedical agents to biodegradable plastics from renewable resources. In spite of the plethora of applications, the large-scale introduction of biopolymers into the market has often been forestalled by high production costs mainly due to complex or inefficient downstream processing. In this article, state-of-the-art methods and recent advances in the separation and purification of microbial polymers are reviewed, with special focus on the biopolymers, γ-polyglutamic acid and xanthan gum. Furthermore, a study of the general factors affecting production and purification is presented, including biopolymer rheology, enzymatic degradation and production of biopolymer mixtures.
 
Bio-water saving can be defined as the reduction of crop water consumption employing biological measures. This is the focus of efforts to save water in agriculture. Different levels of water-use efficiency (WUE) have been developed. The genetic diversity of WUE has been confirmed in several crops. WUE is the basis of bio-watering and physiological WUE is the key. The degree to develop physiological WUE potential decides the performance of bio-watering in the field. During this process, fine management is important. Thus bio-watering is closely related to WUE. Crop WUE has improved and evolved as a result of breeding programs. Many WUE genes have been located in different genomic and aneuploid materials and have been mapped by various molecular markers in a number of crops. Two genes, (Erecta and alx8), which control water use efficiency; have been cloned in Arabidopsis thaliana. Eleven WUE genes have been identified by microarray analysis. Six genes associated with drought resistance and photosynthesis have been transfered into crops which have resulted in improving WUE and drought resistance. WUE is important on the basis of functional identification of more drought resistant gene resources. The popularity on the industrial-scale of transgenic plants is still in its infancy and one of the reasons for this is the lack of knowledge regarding molecular mechanisms and it is a very immature technology. Enhanced agricultural practices and the theoretical aspects of improving crop WUE have been developed and are discussed in this review paper. Rapid progress will be made in bio-water savings and that crop WUE can be substantially improved under both favorable and unfavorable water-limited environments. This will be achieved by a combination of traditional breeding techniques and the introduction of modern biotechnology.
 
The life cycle of the insect of the genus Hepialus (a) and the development process of the DCXC (b). 
Fundamental procedure for breeding of Hirsutella sinensis.
Abstract Ophiocordyceps sinensis (syn. Cordyceps sinensis), a traditional Chinese medicine called DongChongXiaCao (DCXC) in Chinese, is well known and has been used in Asia countries since the fifteenth century, and it contains some valuable medicinal component defined by modern pharmacological science. DCXC only appears at high altitudes on the Qinghai-Tibetan Plateau. Consequently, it is difficult to find and harvest. Because of its rarity and medicinal value, DCXC has always been one of the most expensive medicines known. As the price of DCXC has risen in recent years, thousands of migrants have entered into the various grasslands to search for them in season, which makes ecological environments of the grassland more fragile. In order to relieve the environmental pressures and protect this valuable resource, the artificial cultivation of DCXC involving two aspects of the genus Hepialus and the fungi of the host larvae should be employed and applied at the first available time point. In this article, the reproduction of moth larvae of the genus Hepialus is first described, which includes their ecological characteristics and the methods of artificial feeding. Second, the generation and isolation method of the fungi from DCXC are subsequently summarized, and then the mechanism of fungal spores to attack the moth larvae are restated. Finally, the basic model of artificial cultivation of DCXC is introduced; meanwhile, the potential application of modern biotechnology to the artificial cultivation is analyzed in prospect. This review article will not only expand people's knowledge regarding the artificial cultivation of DCXC, but also hopefully provide an informative reference for the development of this valuable resource and the environmental protection of alpine meadows.
 
Comparison of selected genetic markers employed in plant identification. 
Plant variety and cultivar identification is one of the most important aspects in agricultural systems. The large number of varieties or landraces among crop plants has made it difficult to identify and characterize varieties solely on the basis of morphological characters because they are non stable and originate due to environmental and climatic conditions, and therefore phenotypic plasticity is an outcome of adaptation. To mitigate this, scientists have developed and employed molecular markers, statistical tests and software to identify and characterize the required plant cultivars or varieties for cultivation, breeding programs as well as for cultivar-right-protection. The establishment of genome and transcriptome sequencing projects for many crops has led to generation of a huge wealth of sequence information that could find much use in identification of plants and their varieties. We review the current status of plant variety and cultivar identification, where an attempt has been made to describe the different strategies available for plant identification. We have found that despite the availability of methods and suitable markers for a wide range of crops, there is dearth of simple ways of making both morphological descriptors and molecular markers easy, referable and practical to use although there are ongoing attempts at making this possible. Certain limitations present a number of challenges for the development and utilization of modern scientific methods in variety or cultivar identification in many important crops.
 
In recent years, the advances in microbiology show that biofilms are structurally complex, dynamic and adaptable systems including attributes of multicellular organisms and miscellaneous ecosystems. One may distinguish between beneficial and harmful biofilms appearing in daily life as well as various industrial processes. In order to advance the growth of the former or prevent the latter type of biofilm, a detailed understanding of its properties is indispensable. Besides microbiological aspects, this concerns the determination of mechanical characteristics, which provides the basis for material modelling. In the present paper the existing experimental methods that have been proposed since the 1980s are reviewed and critically discussed with respect to their usefulness and applicability to develop numerical modelling approaches.
 
Different types of enzyme-catalyzed processes are reviewed, with particular regard to those procedures leading to the generation of chiral compounds of high optical purity. The main body of the review deals with hydrolyses and esterification as well as the reduction and oxidation of organic substrates. Other biotransformations of current and/or future importance in the synthesis of homochiral fine chemicals (such as the formation of carbon-carbon bonds using aldolases) are also discussed in some detail. Attention is drawn to current trends in the area and, to this end, a majority of the references are taken from journals published during the period April 1987 to September 1988.
 
Abstract Populations of bacterial cells that grow under the same conditions and/or environments are often considered to be uniform and thus can be described by ensemble average values of their physiologic, phenotypic, genotypic or other parameters. However, recent evidence suggests that cell-to-cell differences at the gene expression level could be an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression or transcriptional-level heterogeneity determines not only the fate of individual bacterial cells in a population but could also affect the ultimate fate of the population itself. Although techniques for single-cell gene expression measurement in eukaryotic cells have been successfully implemented for a decade or so, they have only recently become available for single bacterial cells. This is due to the difficulty of efficient lysis of most bacterial cells, as well as short half-life time (low stability) of bacterial mRNA. In this article, we review the recent progress and challenges associated with analyzing gene expression levels in single bacterial cells using various semi-quantitative and quantitative methods. In addition, a review of the recent progress in applying microfluidic devices to isolate single bacterial cells for gene expression analysis is also included.
 
Many reporter genes, such as gfp, gusA, and lacZ, are widely used for research into plants, animals, and microorganisms. Reporter genes, which offer high levels of sensitivity and convenience of detection, have been utilized in transgenic technology, promoter analysis, drug screening, and other areas. Directed molecular evolution is a powerful molecular tool for the creation of designer proteins for industrial and research applications, including studies of protein structure and function. Directed molecular evolution is based mainly on in vitro recombination methods, such as error-prone PCR and DNA shuffling. The strategies of directed evolution of enzyme biocatalysts have been the subject of several recent reviews. Here, we briefly summarize successes in the field of directed molecular evolution of reporter genes and discuss some of the applications.
 
Aerobic granules developed with added Ca 2+ (left) and 
Pilot-scale (1 m 3 ) SBR with aerobic granules for 
Aerobic granular sludge can be classified as a type of self-immobilized microbial consortium, consisting mainly of aerobic and facultative bacteria and is distinct from anaerobic granular methanogenic sludge. Aerobic granular technology has been proposed as a promising technology for wastewater treatment, but is not yet established as a large-scale application. Aerobic granules have been cultured mainly in sequenced batch reactors (SBR) under hydraulic selection pressure. The factors influencing aerobic granulation, granulation mechanisms, microbial communities and the potential applications for the treatment of various wastewaters have been studied comprehensively on the laboratory-scale. Aerobic granular sludge has shown a potential for nitrogen removal, but is less competitive for the high strength organic wastewater treatments. This technology has been developed from the laboratory-scale to pilot scale applications, but with limited and unpublished full-scale applications for municipal wastewater treatment. The future needs and limitations for aerobic granular technology are discussed.
 
Lycopene is the pigment principally responsible for the characteristic deep-red color of ripe tomato fruits and tomato products. It has attracted attention due to its biological and physicochemical properties, especially related to its effects as a natural antioxidant. Although it has no provitamin A activity, lycopene does exhibit a physical quenching rate constant with singlet oxygen almost twice as high as that of beta-carotene. This makes its presence in the diet of considerable interest. Increasing clinical evidence supports the role of lycopene as a micronutrient with important health benefits, because it appears to provide protection against a broad range of epithelial cancers. Tomatoes and related tomato products are the major source of lycopene compounds, and are also considered an important source of carotenoids in the human diet. Undesirable degradation of lycopene not only affects the sensory quality of the final products, but also the health benefit of tomato-based foods for the human body. Lycopene in fresh tomato fruits occurs essentially in the all-trans configuration. The main causes of tomato lycopene degradation during processing are isomerization and oxidation. Isomerization converts all-trans isomers to cis-isomers due to additional energy input and results in an unstable, energy-rich station. Determination of the degree of lycopene isomerization during processing would provide a measure of the potential health benefits of tomato-based foods. Thermal processing (bleaching, retorting, and freezing processes) generally cause some loss of lycopene in tomato-based foods. Heat induces isomerization of the all-trans to cis forms. The cis-isomers increase with temperature and processing time. In general, dehydrated and powdered tomatoes have poor lycopene stability unless carefully processed and promptly placed in a hermetically sealed and inert atmosphere for storage. A significant increase in the cis-isomers with a simultaneous decrease in the all-trans isomers can be observed in the dehydrated tomato samples using the different dehydration methods. Frozen foods and heat-sterilized foods exhibit excellent lycopene stability throughout their normal temperature storage shelf life. Lycopene bioavailability (absorption) can be influenced by many factors. The bioavailability of cis-isomers in food is higher than that of all-trans isomers. Lycopene bioavailability in processed tomato products is higher than in unprocessed fresh tomatoes. The composition and structure of the food also have an impact on the bioavailability of lycopene and may affect the release of lycopene from the tomato tissue matrix. Food processing may improve lycopene bioavailability by breaking down cell walls, which weakens the bonding forces between lycopene and tissue matrix, thus making lycopene more accessible and enhancing the cis-isomerization. More information on lycopene bioavailability, however, is needed. The pharmacokinetic properties of lycopene remain particularly poorly understood. Further research on the bioavalability, pharmacology, biochemistry, and physiology must be done to reveal the mechanism of lycopene in human diet, and the in vivo metabolism of lycopene. Consumer demand for healthy food products provides an opportunity to develop lycopene-rich food as new functional foods, as well as food-grade and pharmaceutical-grade lycopene as new nutraceutical products. An industrial scale, environmentally friendly lycopene extraction and purification procedure with minimal loss of bioactivities is highly desirable for the foods, feed, cosmetic, and pharmaceutical industries. High-quality lycopene products that meet food safety regulations will offer potential benefits to the food industry.
 
This article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by Savitri and Azmi (2003), our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.
 
Epidemiological data show that a diet rich in fruits and vegetables can reduce the risk from a number of cancers and chronic diseases. Sulforaphane (SF), a phytochemical constituent of cruciferous vegetables, has been widely researched in recent decades as a potential chemopreventive compound. Nonexistent in intact vegetables, natural SF, is formed from glucoraphanin hydrolyzed by myrosinase. This review summarizes and compares different analysis, isolation and purification methods engaged in SF research. Major important chemopreventive properties of SF investigated in existing research are reviewed and discussed, including antioxidant, anticarcinogenic and anti-inflammatory functions. Considering the potential applications of SF in the future, metabolism, stability and formulation developments of SF are also discussed. Research opportunities are identified based on the review of existing studies to facilitate future explorations on SF, a promising natural compound in chemopreventive therapy.
 
Abstract Cross-linked enzyme aggregate (CLEA) technology has been regarded as an effective carrier-free immobilization method. This method is very attractive due to its simplicity and robustness, as well as for the possibility of using the crude enzyme extract and the opportunity to co-immobilize multiple different enzymes. The resulting CLEAs generally exhibit high catalyst productivities, improved storage and operational stability and are easy to recycle. Nowadays, although the technology has been applied to various enzymes, some undesirable properties have limited its further application. To overcome these limitations, novel strategies have been developing in recent years. This mini-review focuses on process optimization, new improved strategies and the latest advances on CLEAs technology.
 
Control of the foodborne pathogens Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes during sufu fermentation was evaluated. Before fermentation, pathogens were inoculated onto tofu (substrate for sufu) at 5 log cfu/g or 3 log cfu/g, and starter culture (Actinomucor elegans) was inoculated at 3 log cfu/g. After 2 days of fermentation at 30 degrees C, the four pathogens reached 7 to 9 log cfu/g, and the mold count reached 6 to 7 log cfu/g. After fermentation, sufu samples were aged in a solution of 10% alcohol + 12% NaCl. After 1 month of aging, the total bacterial count was 6 to 7 log cfu/g, but all foodborne pathogens and mold were reduced to nondetectable levels. The total bacterial count decreased after aging for 2 months and 3 months, but the differences were not significant (P > 0.05) compared with the count after 1 month. Microorganism in experimental sufu from different aging periods and in commercial sufu were compared. A total of 270 isolates were purified and identified by the BBL Crystal Identification System. From the experimental sufu samples, 49 Bacillus spp. (20.4%), 167 Enterococcus spp. (69.6%), 6 Shewanella putrefaciens (2.4%), and 18 miscellaneous gram-negative bacilli (7.5%) were identified. From commercial sufu samples, 17 Bacillus spp. (56.7%), 2 Enterococcus durans (6.7%), 5 miscellaneous gram-negative bacilli (16.7%), 5 Corynbacterium aquaticum (16.7%), and 1 Shewanella putrefaciens (3.3%) were obtained. Although the longer aging period did not significantly decrease the total bacterial count, it may help in the development of sufu flavor. This study showed that sufu fermentation and aging can control common foodborne pathogens, so sufu is a safe product even though its preparation does not include pasteurization.
 
Structure of pectic polysaccharide. 
Mode of action of different enzymes on pectin moiety. PME, Pectin methylesterase; PAE, Pectinacetyl esterase; PG, Polygalacturanase; RGL, Rhamnogalacturonanlyasc; RGAE, Rhamnogalacturonan acetyl esterase; RG, Rhamnogalacturonan hydrolase; AF, Arabinofuraosidase; EA, Endo-arabinase; Endo-Gal, Endo-galactanase; bGal, b-Galactosidase.
Pectic polysaccharide composition of agricultural by-products.
Abstract Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and for in vivo and in vitro studies of important POS, so that they could be commercially exploited.
 
Biofertilizers, namely Rhizobium and biocontrol agents such as Pseudomonas and Trichoderma have been well established in the field of agricultural practices for many decades. Nevertheless, research is still going on in the field of inoculant production to find methods to improve advanced formulation and application in fields. Conventionally used solid and liquid formulations encompass several problems with respect to the low viability of microorganisms during storage and field application. There is also lack of knowledge regarding the best carrier in conventional formulations. Immobilization of microorganisms however improves their shelf-life and field efficacy. In this context, microencapsulation is an advanced technology which has the possibility to overcome the drawbacks of other formulations, results in extended shelf-life, and controlled microbial release from formulations enhancing their application efficacy. This review discusses different microencapsulation technologies including the production strategies and application thereof in agricultural practices.
 
Cytokinins are master regulators of plant growth and development. They are involved in the regulation of many important physiological and metabolic processes. Recent progress in cytokinin research at the molecular level, including identification of related genes and cytokinin receptors, plus elucidation of signal transduction, has greatly increased our understanding of cytokinin actions. Although still in its infant stage, molecular breeding of crops with altered cytokinin metabolism, when combined with the transgenic approach, has shown very promising potential for application to agriculture. In this review we briefly introduce recent progress in cytokinin molecular biology, discuss applications of cytokinin genetic engineering to agriculture, and present implications and future research directions.
 
Top-cited authors
Parvaiz Ahmad
  • GDC Pulwama J&K INDIA/ King Saud University Riyadh Saudi Arabia
Gowher Nabi
  • Jazan University
Satyawati Sharma
  • Indian Institute of Technology Delhi
Hongbo Shao
  • Jiangsu Academy of Agricultural Sciences
Mausam Verma
  • Institut de recherche et de développement en agroenvironnement