In Geol Choi

Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes

Rice straw was pretreated using aqueous-ammonia solution at moderate temperatures to enable production of the maximum amount of fermentable sugars from enzymatic hydrolysis. The effects of various operating variables including pretreatment temperature, pretreatment time, the concentration of ammonia and the solid-to-liquid ratio on the degree of lignin removal and the enzymatic digestibility were optimized using response surface methodology. The optimal reaction conditions, which resulted in an enzymatic digestibility of 71.1%, were found to be 69 degrees C, 10h and an ammonia concentration of 21% (w/w). The effects of different commercial cellulases and the additional effect of a non-cellulolytic enzyme, xylanase, were also evaluated. Additionally, simultaneous saccharification and fermentation was conducted with rice straw to assess the ethanol production yield and productivity.

Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment

Rice straw was irradiated using an electron beam at currents and then hydrolyzed with cellulase and beta-glucosidase to produce glucose. The pretreatment by electron beam irradiation (EBI) was found to significantly increase the enzyme digestibility of rice straw. Specifically, when rice straw that was pretreated by EBI at 80 kGy at 0.12 mA and 1 MeV was hydrolyzed with 60 FPU of cellulase and 30 CBU of beta-glucosidase, the glucose yield after 132 h of hydrolysis was 52.1% of theoretical maximum. This value was significantly higher than the 22.6% that was obtained when untreated rice straw was used. In addition, SEM analysis of pretreated rice straw revealed that EBI caused apparent damage to the surface of the rice straw. Furthermore, EBI pretreatment was found to increase the crystalline portion of the rice straw. Finally, the crystallinity and enzyme digestibility were found to be strongly correlated between rice straw samples that were pretreated by EBI under different conditions.

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482

Global extent of horizontal gene transfer

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. There have been many convincing evidences for HGT for specific genes or gene families, but there has been no estimate of the global extent of HGT. Here, we present a method of identifying HGT events within a given protein family and estimate the global extent of HGT in all curated protein domain families (≈8,000) listed in the Pfam database. The results suggest four conclusions: (i) for all protein domain families in Pfam, the fixation of genes horizontally transferred is not a rampant phenomenon between organisms with substantial phylogenetic separations (1.1–9.7% of Pfam families surveyed at three taxonomic ranges studied show indication of HGT); (ii) however, at the level of domains, >50% of Archaea have one or more protein domains acquired by HGT, and nearly 30–50% of Bacteria did the same when examined at three taxonomic ranges. But, the equivalent value for Eukarya is <10%; (iii) HGT will have very little impact in the construction of organism phylogeny, when the construction methods use whole genomes, large numbers of common genes, or SSU rRNAs; and (iv) there appears to be no strong preference of HGT for protein families of particular cellular or molecular functions.

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353.

Modification of mesenchymal stem cells for cardiac regeneration

Importance of the field: Mesenchymal stem cells (MSCs) have the greatest potential for use in cell-based therapy of human heart diseases, especially in myocardial infarcts. The therapeutic potential of MSCs in myocardial repair is based on the ability of MSCs to directly differentiate into cardiac tissue and on the paracrine actions of factors released from MSCs. However, the major obstacle in the clinical application of MSC-based therapy is the poor viability of the transplanted cells due to harsh microenvironments like ischemia, inflammation and/or anoikis in the infarcted myocardium. Recently, various approaches have been implemented in an effort to improve the survival of implanted MSCs through ex vivo manipulation of MSCs. Areas covered in this review: Major obstacles in MSC-based therapy are discussed, along with recent advances for enhancing therapeutic potential of engrafted MSCs from the past decade. What the reader will gain: This review focuses primarily on ex vivo manipulation of MSCs before transplantation, which includes pretreatment, preconditioning and genetic modification of MSCs, and future directions. Take home message: Modification of MSCs before transplantation has developed into a promising option for enhancing the beneficial effects of MSC-based therapy for cardiac repair after myocardial infarction.

Exploring the human diseasome: the human disease network.

Advances in genome-scale molecular biology and molecular genetics have greatly elevated our knowledge on the basic components of human biology and diseases. At the same time, the importance of cellular networks between those biological components is increasingly appreciated. Built upon these recent technological and conceptual advances, a new discipline called the network medicine, an approach to understand human diseases from a network point-of-view, is about to emerge. In this review article, we will survey some recent endeavours along this direction, centred on the concept and applications of the human diseasome and the human disease network. Questions, and partial answers thereof, such as how the connectivity between molecular parts translates into the relationships between the related disorders on a global scale and how central the disease-causing genetic components are in the cellular network, will be discussed. The use of the diseasome in combination with various interactome networks and other disease-related factors is also reviewed.

Evolution of protein structural classes and protein sequence families

In protein structure space, protein structures cluster into four elongated regions when mapped based solely on similarity among the 3D structures. These four regions correspond to the four major classes of present-day proteins defined by the contents of secondary structure types and their topological arrangement. Evolution of and restriction to these four classes suggest that, in most cases, the evolution of genes may have been constrained or selected to those genetic changes that results in structurally stable proteins occupying one of the four “allowed” regions of the protein structure space, “structural selection,” an important component of natural selection in gene evolution. Our studies on tracing the “common structural ancestor” for each protein sequence family of known structure suggest that: (i) recently emerged proteins belong mostly to three classes; (ii) the proteins that emerged earlier evolved to gain a new class; and (iii) the proteins that emerged earliest evolved to become the present-day proteins in the four major classes, with the fourth-class proteins becoming the most dominant population. Furthermore, our studies also show that not all present-day proteins evolved from one single set of proteins in the last common ancestral organism, but new common ancestral proteins were “born” at different evolutionary times, not traceable to one or two ancestral proteins: “the multiple birth model” for the evolution of protein sequence families.

An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity

Molecular function of the expansin superfamily has been highlighted for cellulosic biomass conversion. In this report, we identified a new bacterial expansin subfamily by analysis of related bacterial sequences and biochemically examined a member of this new subfamily from Hahella chejuensis (HcEXLX2). Among the various complex polysaccharides tested, HcEXLX2 bound most efficiently to cellulose. The relative binding constant (Kr) against Avicel was 2.1 L g−1 at pH 6.0 and 4°C. HcEXLX2 enhanced the activity of cellulase, producing about 4.6 times more hydrolysis product after a 36 h reaction relative to when only cellulase was used. The extension strength test on filter paper indicated that HcEXLX2 has a texture loosening effect on filter paper, which was 53% of that observed for 8 M urea treatment. These activities, compared with a cellulose binding domain from Clostridium thermocellum, implied that the synergistic effect of HcEXLX2 comes from not only binding to cellulose but also disrupting the hydrogen bonds in cellulose. Based on these results, we suggest that the new bacterial expansin subfamily functions by binding to cell wall polysaccharides and increasing the accessibility of cell wall degrading enzymes.

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators’ laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1−6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6−10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.