Pectinase production by a Brazilian thermophilic fungus <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> N31 in solid-state and submerged fermentation

Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45°C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1 : 1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.     

Biodegradation and detoxification of dyes and industrial effluents by Ganoderma weberianum B-18 immobilized in a lab-scale packed-bed bioreactor

White-rot fungi are considered to be promising biotechnological tools to complement or replace the current technologies for the treatment of effluents from textile production plants. The aim of this work was to investigate the decolorization capacity of Ganoderma weberianum B-18 in solid state fermentation with sugarcane bagasse as a substrate and ligninolytic inducer as well as to decolorize and detoxify industrial effluents by this strain in a laboratory scale packed-bed bio-reactor. The results demonstrated that G. weberianum B-18 indeed showed to possess decolorization capacity in solid state fermentation with sugarcane bagasse supplemented with synthetic dyes. Moreover, fungal biomass of G. weberianum B-18 immobilized in sugarcane bagasse in a packed-bed bioreactor was shown to efficiently decolorize and detoxify different dyes and authentic industrial effluents in semi-continuous conditions. In this decolorization process, laccase enzymes secreted by the fungus played the main role. Hence, a packed-bed reactor with G. weberianum B-18 immobilized in sugarcane bagasse seems to be a suitable system for the further development of an efficient bioprocess for large-scale treatment of dye-containing wastewaters.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

草酸青霉的植物多糖降解酶基因表达调控的研究进展

植物生物质是地球上最丰富的可再生资源,对其生物炼制可生产高附加值的生物基产品。生物炼制需要使用植物多糖降解酶(plant-polysaccharide-degrading enzymes,PPDEs),如纤维素酶、木聚糖酶和生淀粉酶。丝状真菌草酸青霉(Penicillium oxalicum)能分泌完整的具有高活力的植物多糖降解酶,但其产量低限制了大规模生产及应用。草酸青霉中植物多糖降解酶的生物合成受到多种调控因子包括转录因子的严格调控。本文主要介绍在以植物生物质甘蔗渣和木薯生淀粉为原料的生物炼制中,涉及的一些关键微生物方面的问题,如从高产植物多糖降解酶的真菌菌株的筛选、育种,到草酸青霉植物多糖降解酶合成及其基因表达的调控基因的鉴定,以及酶产量提高的工程菌株的构建等,为丝状真菌资源的开发与利用提供理论指导。     

Efficacy of Paecilomyces lilacinus (strain 251) for the control of Radopholus similis in banana

Paecilomyces lilacinus is a common soil fungus that has been isolated from many different habitats around the world. It is well known as a facultative egg pathogen of sedentary nematodes and also an important option to control Radopholus similis juvenile and adults in banana. This nematode antagonistic fungus may be used in an integrated approach to control banana plant parasitic nematodes. Dose response and form of application experiments were conducted with burrowing nematode, R. similis, on banana using a commercial water dispersible granulate formulated P. lilacinus (strain 251) product. The results revealed that nematode activity decreased in the presence of this fungus. An important correlation between rates of application and the degree of control of R. simnilis penetration and banana root weight was observed. The best control was achieved in the treatment were plantlets and soil were pre-inoculated with P. lilacinus and reinoculated during transplantation. The results showed that the biocontrol agent P. lilacinus is an excellent candidate for an IPM program against nematodes such as Radopholus similis.     

Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in <Emphasis Type="Italic">Phlebia tremellosa</Emphasis>

A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.     

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.     

Role of Rhizomys pruinosus as a natural animal host of Penicillium marneffei in Guangdong,China

Penicillium marneffei, a dimorphic fungus that can cause penicilliosis marneffei, is endemic in Southeast Asia. The only known hosts of P. marneffei are humans and bamboo rats. The aim of our study was to explore the distribution of P. marneffei in bamboo rats, their associated environment and non‐rat‐associated environments. Totally, 270 samples were collected in Guangdong province of China in 2012; the prevalence of P. marneffei was much higher in samples collected from surrounding areas of burrows (8.2%) than in the samples obtained from non‐rat‐associated sites (2%) or artificial farms of bamboo rats (0%). There was no difference in P. marneffei isolated rate from different areas of Guangdong province. The infection is prevalent in all rats, and this fungus could be frequently seen in the rats' lungs. This study confirms that bamboo rat is the ecological niche of P. marneffei and hypothesizes that bamboo rats become infected by inhaling aerosolized conidia originating from environmental sources, rather than by the fecal–oral route or transplacental crossing. According to the result of no detection of P. marneffei in the artificial farm, the activity of bamboo rats might be more relevant to the distribution and dissemination of P. marneffei in natural environment.     

An alternative, banana peel-based medium used to investigate the catalytic properties of peroxidase from a fungus, Inonotus sp SP2, recently isolated in southern Chile

Recycling industrial wastes is one of the major goals of bioengineering research. Agricultural wastes are often rich in natural sources of organic and inorganic compounds. The present study investigates the use of banana peel waste as a non-conventional alternative to nitrogen-enriched glucose media for a white rot fungus (WRF), Inonotus sp. SP2, recently isolated in southern Chile. WRF are known to produce biodegrading enzymes, such as peroxidases, that can have industrial and biotechnological applications. To that end, the metabolic characteristics and catalytic properties of peroxidases produced by Inonotus sp. SP2 were compared between glucose and banana peel-based growth mediums. The results establish that this strain of WRF produces high concentrations of a Mn⁺²-dependent peroxidase, with greater enzymatic activity in extracellular fluid and crude enzyme extracted from fungus grown in banana peel and glucose media, respectively. H₂O₂ has an inhibiting effect that is greater for enzymes produced in glucose media, and greater biomass can be obtained in banana-peel based media. This demonstrates that banana peel is a suitable and more cost-effective alternative to conventional glucose-based media for the production of biodegradative enzymes, such as peroxidase. Unlike other strains of WRF, the metabolic characteristics of Inonotus sp. SP2 demonstrate that it enters secondary metabolism with the production oxidative enzymes after both carbon and nitrogen sources are depleted. This suggests that with further investigation, this strain of WRF may be useful in industrial applications that require the biodegradation of nitrogen and carbon-based wastes and recalcitrant compounds.     

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification.     

四种添加物对铁皮石斛原球茎生长及多糖含量的影响

为探讨铁皮石斛(Dendrobium officinale)培养基中添加物的作用,在1/2MS培养基中加入椰肉、甘蔗渣、香蕉皮和麦麸等4种添加物,研究不同浓度添加物和培养时间对原球茎生长和多糖含量的影响。结果表明,4种添加物对铁皮石斛原球茎的增殖、分化和多糖含量均有一定影响,其中添加15.0 g L–1甘蔗渣,培养60 d能明显促进铁皮石斛原球茎的增殖与分化(146.1%);而添加20.0 g L–1甘蔗渣,培养40 d能显著提高铁皮石斛原球茎多糖含量(50.4%)。这说明甘蔗渣是培养铁皮石斛原球茎的适宜添加物,既能促进铁皮石斛原球茎的生长发育,还能降低生产成本。     

A new strain of <Emphasis Type="Italic">Bjerkandera</Emphasis> sp. production,purification and characterization of versatile peroxidase

The lignin modifying enzymes (LMEs) secreted by a new white rot fungus isolated from Chile were studied in this work. This fungus has been identified as a new anamorph of Bjerkandera sp. based on the sequences of the ribosomal DNA and morphological analysis at light microscopy showing hyaline hyphae without clamp connection, cylindrical conidia and lack of sexual forms, similar to those reported in other Bjerkandera anamorphs. The characterization of the culture medium for the highest LMEs production was performed in flask cultures, with a formulation of the culture medium containing high levels of glucose and peptone. The highest Mn-oxidizing peroxidase activity (1,400 U/L) was achieved on day 6 in Erlenmeyer flasks. Four peroxidases (named R1B1, R1B2, R1B3 and R1B4), have been purified by using ion-exchange and exclusion molar chromatographies. All of them showed typical activity on Mn²⁺ and exhibited Mn-independent activity against 2,6-dimethoxyphenol. R1B4 showed also activity on veratryl alcohol (pH 3) indicating that this enzyme belongs to the versatile peroxidase family. The high VP production capacities of this strain, as well as the enzymatic characteristics of the LMEs suggest that it may be successfully used in the degradation of recalcitrant compounds.     

Isolation of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> From Soil and Wild Rodents in Guangdong,SE China

Infection by Penicillium marneffei is an important emerging public health problem, especially among travelers and inhabitants in SE China and SE Asia infected with human immunodeficiency virus (HIV). In recent years, the number of patients with penicilliosis marneffei (PM) has increased rapidly in Guangdong province, SE China. However, the natural habitat and transmission mode of the etiologic agent remains unclear. In this study, wild rats (Microtus, focus Rattus and Rhizomys pruinosus) and soil samples were collected from rat burrows, populated and rural areas from November 2007 to December 2008 for fungus cultures. All isolates, suspected of being P. marneffei, were identified by gross and microscopic morphology and ITS analysis. Sixteen of 23 (about 70%) bamboo rats were P. marneffei positive, whereas none was recovered from hamsters, loirs or soil. This suggests that as of today the bamboo rat is the exclusive natural reservoir for P. marneffei. Definite evidence that rodents are a part of the infectious cycle is still lacking.     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.     

An experimental study of bamboo ants in western Amazonia

Numerous ant taxa naturally inhabit stems of live and dead Guadua bamboo (Bambusoidea, Poaceae) in western Amazonia. In an experiment at the onset of the wet season in Peru’s Manu National Park, we augmented potential nest sites in stems of live bamboo, dead bamboo and dead ca?a brava (Gynerium sagittatum, another woody grass) at five stations within each of ten bamboo patches and ten control areas outside those patches. Each experimental stem possessed three vacant and available internodes, pre-drilled with, respectively, large, small and linear holes, mimicking the range of forms of surveyed natural entrances. After 24 days, approximately 13% of 798 available internodes had been colonized, the majority by fragments of existing colonies. Ignoring entrance type, which did not affect colonization for any species or species group, and censoring non-independent internodes of the same stem, we used individual stems as independent sample units in other tests. One specialist in live bamboo (Camponotus longipilis), and a likely specialist in dead bamboo (Camponotus depressus), were identified based on overrepresentation in bamboo habitat and disproportionate occurrence in live or dead bamboo stems. A third species, Camponotus (Pseudocolobopsis sp.) was more abundant in bamboo areas but colonized both dead bamboo and dead ca?a. Relatively high abundance of standing dead stems in Guadua forests may account for the presence of a dead stem specialist. The experiment missed detecting specialization in one live culm specialist (Camponotus mirabilis), likely due to its failure to simulate conditions required for the species’ unique modes of colony establishment and spread into new culms. Most opportunistic stem nesters colonized dead bamboo at significantly greater rates than dead ca?a, but were either equally well represented in bamboo and control areas, or underrepresented in bamboo habitat. Given low colonization rates overall, underrepresentation in bamboo cannot be attributed to competition from bamboo specialists for nesting space. Rather, it may be due to combined effects of seasonal flooding of bamboo habitat, and greater importance of food limitation, relative to nest site limitation, in that habitat. Received 9 May 2005; revised 25 August 2005; accepted 29 August 2005.     

Defoliation effects on enzyme activities of the ectomycorrhizal fungus <Emphasis Type="Italic">Suillus granulatus</Emphasis> in a <Emphasis Type="Italic">Pinus contorta</Emphasis> (lodgepole pine) stand in Yellowstone National Park

Ectomycorrhizal (EM) basidiomycete fungi are obligate mutualists of pines and hardwoods that receive fixed C from the host tree. Though they often share most recent common ancestors with wood-rotting fungi, it is unclear to what extent EM fungi retain the ability to express enzymes that break down woody substrates. In this study, we tested the hypothesis that the dominant EM fungus in a pure pine system retains the ability to produce enzymes that break down woody substrates in a natural setting, and that this ability is inducible by reduction of host photosynthetic potential via partial defoliation. To achieve this, pines in replicate blocks were defoliated 50% by needle removal, and enzyme activities were measured in individual EM root tips that had been treated with antibiotics to prevent possible bacterial activity. Results indicate that the dominant EM fungal species (Suillus granulatus) expressed all enzymes tested (endocellulase D-glucosidase, laccase, manganese peroxidase, lignin peroxidase, phosphatase and protease), and that activities of these enzymes increased significantly (P < 0.001) in response to defoliation. Thus, this EM fungus (one of the more specialized mutualists of pine) has the potential to play a significant role in C, N and P cycling in this forested ecosystem. Therefore, many above-ground factors that reduce photosynthetic potential or divert fixed C from roots may have wide-reaching ecosystem effects.     

<Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var.<Emphasis Type="Italic"> rhizopodiformis</Emphasis>: a thermotolerant fungus with potential for production of thermostable amylases

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45°C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of -amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.     

Differential expression of pathogenesis-related proteins and defense enzymes in banana: interaction between endophytic bacteria,Banana bunchy top virus and Pentalonia nigronervosa

Banana bunchy top disease caused by Banana bunchy top virus is the most serious viral disease of banana and plantain worldwide. The virus is transmitted by the aphid vector Pentalonia nigronervosa in a persistent manner. This paper deals with the effect of the interaction between plant growth promoting endophytic bacteria, Banana bunchy top virus, and the banana aphid Pentalonia nigronervosa in the expression of Pathogenesis-related proteins (PR-proteins) and defense enzymes in banana. The existence of virus in the aphids was confirmed by ELISA, DIBA and PCR. PCR could amplify 1100-bp replicase gene of BBTV from viruliferous aphids. A significant increase in the enzymatic activity of all measured PR proteins and defense enzymes, as compared to control plants, was seen in the plants inoculated with endophytic bacteria and challenged with viruliferous aphids. Native gel electrophoresis revealed expression of more isoforms of PR proteins viz., peroxidase and chitinase in the banana plants challenged with mixtures of plant growth promoting endophytic bacteria and viruliferous aphids. Enhanced activity of a PR-2 protein viz., β-1,3-glucanase was also noticed in the viruliferous aphids infested plants. Some of the defense-related enzymes viz., Polyphenol oxidase and Phenylalanine ammonia lyase and phenolic compounds were also upregulated, up to 5 days after aphid infestation and thereafter there was a reduction in the enzymatic activity. Thus, there exist a differential accumulation of PR proteins and defense-related enzymes, when there is tri-tropic interaction between endophytic bacteria, virus, and insect and the role of the endophytic bacteria in the defense mechanisms against insect pests needs to be elucidated.     

18份广东香蕉种质对枯萎病的抗性评价

【背景】香蕉枯萎病是世界性的香蕉毁灭性病害,尚无有效药剂防控,筛选抗病品种是目前理想的防治方法。【方法】采用组培苗伤根接种法,研究了18份香蕉种质对香蕉枯萎病菌4号生理小种的抗性水平,并根据病情指数进行抗性分级。【结果】在供试的18份香蕉种质中,2份(东莞大蕉、抗枯5号)高抗,2份(碧盛、大丰)抗病,3份(抗枯1号、粉杂、农科1号)中抗,7份(粤优抗1号、广东-741、泰国B9、大蕉、台湾8号、海贡蕉、威廉斯8818)感病,4份(巴西、广东2号、广粉1号、粉蕉)高感。【结论与意义】不同香蕉种质对香蕉枯萎病菌4号生理小种的抗病性存在较大差异,本研究初步筛选出7份抗枯萎病的香蕉种质,为香蕉枯萎病抗病育种提供了依据,为病区种植香蕉品种提供了有效参考。     

Extracellular enzyme production byThermomonospora curvata grown on bagasse

Summary Extracellular enzyme production by the actinomycete,Thermomonospora curvata, was characterized during growth at 55°C on bagasse as sole carbon source. Mycelia adhered to the bagasse fibers during early growth and were released in mature cultures. Extracellular protein reached a maximum on 4% (w/v) bagasse and yielded an electrophoretic profile similar to those produced on purified cellulose. Cellulase production on bagasse exceeded that observed forT. curvata on any previously employed substrate. Amylase and pectinase, which were diminished by their instability in culture fluid at growth temperature and by the lack of inducing substrate, were readily inducible by addition of starch or pectin, respectively. Extracellular activities of -glucosidase and -xylosidase remained insignificant throughout growth. Xylanase production equaled or exceeded that observed on a variety of other substrates. The combined activity of extracellular enzymes from bagasse-grownT. curvata caused a 27% solubilization of the fiber, yielding a mixture of cellooligosaccharides, cellobiose, xylobiose, glucose, xylose, fructose, arabinose and mannitol. Fractionation of concentrated extracellular proteins by size exclusion chromatography yielded single peaks for amylase and pectinase (estimated molecular weights of 58 K and 34 K respectively), while cellulase and xylanase activities were distributed throughout a series of multiple unresolved peaks spanning a molecular weight range of 26–180 K.