Isolation of tannase‐producing microbiota from the gastrointestinal tracts of some freshwater fish

Tannins are the most abundant among the plant‐derived antinutrients that bind readily with protein and other macromolecules to form indigestible complexes, thereby reducing the nutritional value of the plant feedstuffs. Presence of tannase‐producing gut microbiota in herbivorous animals has been suggested to overcome the antinutritional effects of tannins. However, this topic has been less investigated in herbivorous/omnivorous fish species. The present study was undertaken to evaluate the presence of tannase‐producing autochthonous microbiota in the gastrointestinal (GI) tracts of some culturable freshwater teleosts and to identify most promising tannase‐producing strains by molecular methods. Isolation and enumeration of tannase‐producing autochthonous microbiota have been carried out in the gut of ten culturable freshwater teleosts, namely catla (Catla catla), silver carp (Hypophthalmichthys molitrix), rohu (Labeo rohita), grass carp (Ctenopharyngodon idella), mrigal (Cirrhinus mrigala), common carp (Cyprinus carpio), bata (Labeo bata), kalbasu (Labeo calbasu), tilapia (Oreochromis mossambicus), and Nile tilapia (Oreochromis niloticus). Culturable heterotrophic and tannase‐producing microbial populations evaluated on tryptone soya agar and selective tannic acid agar media, respectively, revealed the maximum in the hindguts of all fish species studied. Out of 72 tannase‐producing colonies, 18 randomly selected isolates were maintained as pure cultures and evaluated quantitatively for tannase production. Among these, four most promising tannase producers were identified by 16S/26S rDNA sequencing following nucleotide blast and deposited in the National Centre for Biotechnology Information (NCBI) GenBank. The strain LR01 isolated from rohu was a bacterium, Enterobacter asburae (GenBank Accession No. GU939631 ). However, the strains CM02, OM01 and LR03 isolated from mrigal, tilapia and rohu were yeasts and identified as Pichia kudriavzevii (GenBank Accession No. GU939629 ), Candida tropicalis (GenBank Accession No. GU911469 ) and Candida parapsilosis (GenBank Accession No. GU939630 ), respectively. To the authors' knowledge, the present study is the first to report tannase‐producing autochthonous microbiota in the gut of freshwater teleosts. Tannin‐degrading microbiota detected in the present study may endow the fish with some ecological advantages by enabling them to overcome the anti‐nutritional effects of plant tannins.     

Characterization and identification of phytase-producing bacteria isolated from the gastrointestinal tract of four freshwater teleosts

Isolation and enumeration of phytase-producing bacteria in the proximal intestine (PI) and distal intestine (DI) of four freshwater teleosts, Nile tilapia (Oreochromis niloticus), murrel (Channa punctatus), climbing perch (Anabas testudineus), and stinging catfish (Heteropneustes fossilis) have been carried out following enrichment culture technique. The bacterial isolates were screened on the basis of their phytase-producing ability. In modified phytase screening medium (MPSM), phytase-producing strains were recorded at higher densities in the PI of Nile tilapia and climbing perch and at a minimum in the DI of catfish. Out of 32 isolates, 20 phytase-producing strains (9 from the DI and 11 from the PI) were primarily selected on the basis of qualitative assay on MPSM plates. Among these isolates, 3 strains (2 from the PI and 1 from the DI) were selected as potent phytase producers according to quantitative enzyme assay. Maximum phytase activity was detected in the bacterial strain ONF2 isolated from the PI of O. niloticus followed by CPF6 and CPH6, isolated from the PI and DI, respectively of C. punctatus. All the three selected phytase-producing strains were Gram-positive rods, capable of forming endospores, and could tolerate a wide range of temperature (25–42 °C) and pH (6–10). The strain CPF6 was able to grow at temperatures up to 55 °C. On the basis of 16S rDNA sequence analysis, isolates ONF2, CPF6 and CPH6 were identified as Bacillus licheniformis. The strain ONF2 showed 100 % similarity to B. licheniformis strain LCR32 (Accession no. FJ976541.1) whereas CPF6 and CPH6 showed 99 % similarity to B. licheniformis strain LCR32 (Accession no. FJ976541.1).     

Production,characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.     

云南5个地区戟叶酸模花中酵母菌和类酵母的多样性

[目的]研究云南5个地区(晋宁、祥云、程海、泸沽湖、洱海)的戟叶酸模(Rumex hastatus)花中的酵母菌和类酵母.[方法]采用涂布平板法对5个地区的戟叶酸模花中酵母菌和类酵母进行分离,通过26S rDNA Dl/D2区域序列分析并结合形态观察对分离获得的酵母菌和类酵母进行鉴定;采用胞外酶定性筛选培养基进行产酶筛选;用苏丹黑B染色法筛选产油脂菌株.[结果]从戟叶酸模花中分离得到82株酵母菌和99株类酵母;82株酵母菌鉴定为6个属16个种和1个潜在新种,99株类酵母鉴定为短梗霉属(Aureobasidium)的普鲁兰类酵母(A.pullulans)及3个变种;戟叶酸模花中的优势属是类酵母短梗霉属,其次为红酵母属(Rhodotorula)和隐球酵母属(Cryptococcus);筛选到134株具有产胞外酶活性和83株产油脂的酵母菌和类酵母.[结论]研究结果显示5个地区的戟叶酸模花中酵母菌和类酵母种类多样性较为丰富,并具有产淀粉酶、蛋白酶、纤维素酶、脂肪酶和油脂的特点,有潜在的应用前景.     

马蜂肠道菌的分离鉴定及产消化酶功能菌株的筛选

【背景】马蜂(Vespa mandarinia Smith)可以防治多种田间害虫,还具有药用价值,其肠道菌群结构和功能还有待研究。【目的】获得马蜂肠道可培养细菌并筛选出具有产消化酶功能的菌株,为理解肠道菌对宿主的影响机理及功能菌株的利用提供科学依据和研究材料。【方法】采用传统细菌分离培养法获得马蜂肠道菌,结合形态学以及16S rRNA基因序列分析进行鉴定;利用水解圈法分别筛选产蛋白酶、脂肪酶、淀粉酶和纤维素酶菌株;通过测量水解圈D与菌落直径d的比值,比较不同细菌的产酶能力。【结果】在马蜂肠道中共分离出6属10种细菌,其中芽孢杆菌属5种,肠球菌属、葡萄球菌属、明串珠菌属、乳球菌属和不动杆菌属各1种。从获得的61个菌株中筛选出6个具有产消化酶功能的菌株。其中,苏云金芽孢杆菌V44具有产蛋白酶、淀粉酶、脂肪酶和纤维素酶4种消化酶的能力;粪肠球菌V6具有产淀粉酶、蛋白酶和脂肪酶3种消化酶的能力;蜡样芽孢杆菌V43具有产蛋白酶、淀粉酶和纤维素酶3种消化酶的能力;粪肠球菌V20、蜡样芽孢杆菌V19和维德曼氏芽孢杆菌V22均具有产蛋白酶的能力。【结论】马蜂肠道细菌资源较丰富,部分有产消化酶的功能,可帮助马蜂消化食物,对宿主健康有一定影响。本研究筛选的6个菌株都能产蛋白酶,其中菌株V43和V44分别具有最强产淀粉酶和脂肪酶的能力,是可进一步开发利用的肠道功能菌株资源。     

Mutants ofXanthomonas campestris defective in secretion of extracellular enzymes

Summary Mutants ofXanthomonas campestris B 1459 were isolated that are defective in secretion of both cellulase and amylase. Both enzymes accumulated in the periplasmic space. The defects in secretion of cellulase or amylase were partly overcome by introducing into the mutants specific multiple copies of DNA cloned fromX. campestris, and presumed to code for cellulase or amylase enzymes. The mutant strains also showed reduced amounts of extracellular pectinase and protease activities, as if the mutants were generally defective for secretion of extracellular enzymes. The mutants showed reduced pathogenesis for turnip seedlings. The secretion-defective mutants may allow production of xanthan gum with reduced cellulose, pectin, protein and starch-degrading enzyme activities, thereby allowing more widespread mixing of microbially produced xanthan gum with these commercially important water-soluble polymers.     

Isolation and characterization of polymorphic microsatellites in Labeo rohita and their cross‐species amplification in related species

The major Indian carps namely rohu (Labeo rohita), catla (Catla catla), mrigal (Cirrhinus mrigala) and calbasu (Labeo calbasu) are important freshwater species of the Indian subcontinent constituting over 65% of the fish produce. In the present study, isolation of 12 microsatellite loci from rohu has been reported. Cross‐species amplification in related carps and their implication in population genetic studies as well as selective breeding program were discussed.     

Carbohydrases in the digestive tract of the African bony-tongue Heterotis niloticus (Pisces: Osteoglossidae)

The presence of amylase and maltase in the stomach, intestine and pyloric caeca of Heterotis niloticus is demonstrated. Amylase activity was highest in the fore-gut, followed by the pyloric caeca, while the lowest activity was in the hind-gut. Optimum pH for intestinal amylase was 8.45. Sucrase, lactase and cellulase were not detected.     

云南高原湖泊抚仙湖和星云湖的酵母菌胞外酶活性

【背景】高原湖泊因其海拔高、气压低、辐射强、氧气含量低,是一类特殊环境,而其中的微生物是高原湖泊生态系统物质循环与能量流动的重要参与者,其胞外酶活性的表现决定其适应这一特殊环境的方式与能力。【目的】对分离自云南高原湖泊抚仙湖和星云湖湖水的酵母菌进行产胞外酶活性的筛选,以期获得具有潜在应用价值的活性菌株。【方法】在5°C和25°C培养温度下,采用平板筛选法对两个湖泊酵母菌进行产胞外蛋白酶、纤维素酶、淀粉酶、脂肪酶、几丁质酶、木聚糖酶、植酸酶、菊粉酶、漆酶、锰依赖过氧化物酶和木质素过氧化物酶活性的筛选。【结果】抚仙湖和星云湖的所有测试酵母菌菌株至少都能产1种胞外酶,且主要产植酸酶、菊粉酶和淀粉酶;其次为脂肪酶、纤维素酶、木聚糖酶、锰依赖过氧化物酶和木质素过氧化物酶;产几丁质酶、蛋白酶和漆酶的酵母菌很少,星云湖酵母菌都不产漆酶。培养温度为5°C时,抚仙湖和星云湖的酵母菌产5种及5种以上胞外酶的活性菌株数均多于25°C。【结论】抚仙湖和星云湖的酵母菌产胞外酶菌株多样性丰富,胞外酶种类多样,产酶酵母菌可能参与高原湖泊生态系统的物质循环;筛选得到的产胞外酶菌株为开发与利用高原湖泊酶资源提供了良好的种质资源,具有进一步研究的价值。     

Diversity of Yeasts from Puddles in the Vicinity of Midre Lovénbreen Glacier, Arctic and Bioprospecting for Enzymes and Fatty Acids

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C_{18:1 (w9C)} and C_{18:2 (w9,12C)} were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8°C compared to 22°C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.     

朱鹮肠道微生物多样性与产酶活性

【背景】朱鹮是我国国家一级保护动物,属于世界上最濒危的鸟类之一。对朱鹮肠道微生物的多样性和产胞外酶活性进行分析,可为朱鹮种群数量恢复提供思路。【目的】了解朱鹮肠道微生物的多样性,测定其产胞外酶活性。【方法】采用纯培养的方法获得朱鹮肠道微生物,通过革兰氏染色和生理生化鉴定,结合16S rRNA基因扩增和序列分析对细菌进行鉴定。使用水解圈法筛选产淀粉酶、蛋白酶、纤维素酶、脂肪酶的菌株。【结果】从人工喂养的朱鹮新鲜粪便中共分离到296株细菌,共计2个门11个属。变形菌门(Proteobacteria) 236株,占分离总数的79.73%,分别为：埃希氏菌属(Escherichia) 137株,占分离总数的46.28%;哈夫尼亚菌属(Hafnia) 39株,占分离总数的13.18%;变形菌属(Proteus) 28株,占分离总数的9.46%;柠檬酸杆菌属(Citrobacter) 23株,占分离总数的7.77%;气单胞菌属(Aeromonas) 6株,占分离总数的2.03%;肠杆菌属(Enterobacter) 1株,占分离总数的0.34%;志贺菌属(Shigella) 1株,占分离总数的0.34%;克雷伯菌属(Klebsiella) 1株,占分离总数的0.34%。厚壁菌门(Firmicutes) 60株,占分离总数的20.27%,分别为：肠球菌属(Enterococcus) 33株,占分离总数的11.15%;库特氏菌属(Kurthia) 14株,占分离总数的4.73%;芽孢杆菌属(Bacillus) 13株,占分离总数的4.39%。优势菌群为变形菌门(Proteobacteria)中的埃希氏菌属(Escherichia),占细菌总数的46.28%。经过生理生化鉴定,每个菌株生理生化鉴定出的种属与各自的16S rRNA基因鉴定出的种属相一致。产酶活力分析结果显示有238株产蛋白酶、25株产脂肪酶、24株产淀粉酶、15株产纤维素酶,分别占分离总数的80.41%、8.45%、8.11%和5.07%。【结论】朱鹮肠道微生物分离出的细菌可分为2门11属,优势菌群为变形菌门(Proteobacteria)中的埃希氏菌属(Escherichia),占细菌总数的46.28%;产酶活性分析显示,80.41%的菌株具有产蛋白酶能力。     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

4株羊瘤胃功能性细菌的筛选

通过DNS法测定羊瘤胃源功能性细菌产生的纤维素酶和淀粉酶的活力,福林酚法测定产生的蛋白酶的活力,检测细菌产生酶的特性。同时检测菌株的发酵液对大肠埃希菌(ATCC25922)、副溶血弧菌(ATCC17802)、藤黄八叠球菌(HY78)和产气杆菌(AS1489)等指示菌的抑制能力,分析它们的抑菌活性。结果表明,羊瘤胃源细菌C13产生的纤维素酶活力最高,产酶量也最高;而细菌C5产淀粉酶活力和蛋白酶活力最高,产生淀粉酶和蛋白酶的能力也最高。抑菌活性检测发现,细菌C9对副溶血弧菌(ATCC17802)有很高的抑制作用,而细菌C12对大肠埃希菌(ATCC25922)的抑制能力最明显。     

Screening of marine actinobacteria for amylase enzymes inhibitors

Amylase inhibitor producing actinobacteria were isolated and characterized from terrestrial environment and there is no much report found from marine environment, hence in the present study, 17 strains isolated from the rhizosphere sediments of mangroves were tested for their amylase inhibition ability. Seawater requirement test for the growth of actinobacteria found that the strains SSR-3, SSR-12 and SSR-16 requires at least 50% and SSR-6 requires at least 25% seawater for their growth. The inhibition activity of both prokaryotic and eukaryotic amylase was tested by using Bacillus subtilis and Aspergillus niger. The maximum amylase activity (40mm) produced by the A. niger was taken as positive control, when the test actinobacteria strains grown in the medium they inhibited amylase activity and was evidenced by the reduction in inhibition zone (14–37 mm) similarly the amylase produced by the Bacillus subtilis was also recorded maximum (35 mm) amylase activity and was taken as positive control, and the test atinobacterial strains reduced enzyme action(12–33 mm) it varied levals. This indicates that the actinobacteria strains were controlled amylase enzyme activity in both the cases. The strain SSR-10 was highly effective and SSR-8 was less effective in inhibiting eukaryotic amylase produced by A. niger. The strain SSR-2 was effective and SSR-6 showed very less effect in inhibiting the prokaryotic amylase produced by the B subtilis.     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.     

Plant Growth-Promoting Bacteria Facilitate the Growth of Barley and Oats in Salt-Impacted Soil: Implications for Phytoremediation of Saline Soils

Plant growth-promoting bacteria (PGPB) strains that contain the enzyme 1-amino- cyclopropane-1-carboxylate (ACC) deaminase can lower stress ethylene levels and improve plant growth. In this study, ACC deaminase-producing bacteria were isolated from a salt-impacted (～50 dS/m) farm field, and their ability to promote plant growth of barley and oats in saline soil was investigated in pouch assays (1% NaCl), greenhouse trials (9.4 dS/m), and field trials (6–24 dS/m). A mix of previously isolated PGPB strains UW3 (Pseudomonas sp.) and UW4 (P. sp.) was also tested for comparison. Rhizobacterial isolate CMH3 (P. corrugata) and UW3+UW4 partially alleviated plant salt stress in growth pouch assays. In greenhouse trials, CMH3 enhanced root biomass of barley and oats by 200% and 50%, respectively. UW3+UW4, CMH3 and isolate CMH2 also enhanced barley and oat shoot growth by 100%–150%. In field tests, shoot biomass of oats tripled when treated with UW3+UW4 and doubled with CHM3 compared with that of untreated plants. PGPB treatment did not affect salt uptake on a per mass basis; higher plant biomass led to greater salt uptake, resulting in decreased soil salinity. This study demonstrates a method for improving plant growth in marginal saline soils. Associated implications for salt remediation are discussed.     

杜比亚蟑螂肠道菌的分离鉴定及产消化酶功能菌株的筛选

【背景】杜比亚蟑螂(Blaptica dubia)可用于活体饲料、化妆品和医药保健品的生产,其肠道菌的研究对杜比亚蟑螂的饲养和肠道菌资源的开发与利用都十分重要。【目的】揭示杜比亚蟑螂肠道可培养菌的种类,筛选具有产消化酶功能的菌株,为理解肠道菌对宿主的影响机理及功能菌株的利用提供科学依据和研究材料。【方法】采用体外培养法获得杜比亚蟑螂肠道菌,结合形态学和分子生物学方法进行鉴定;用水解圈法分别筛选产纤维素酶、蛋白酶、脂肪酶和淀粉酶菌株。【结果】在杜比亚蟑螂肠道中共分离出4属7种细菌,其中芽孢杆菌属(Bacillus)2种,沙雷氏菌属(Serratia)和柠檬酸杆菌属(Citrobacter)各2种,肠球菌属(Enterococcus)1种。从获得的20个菌株中筛选出10个具有产消化酶功能的菌株。其中,芽孢杆菌属的菌株D6、D12和D20具有产纤维素酶、蛋白酶、淀粉酶及脂肪酶4种消化酶的功能;沙雷氏菌属的菌株D3、D7、D9、D11和D15具有产纤维素酶、蛋白酶和脂肪酶3种消化酶的能力;柠檬酸杆菌属的菌株D5具有产纤维素酶的功能;肠球菌属的菌株D17具有产蛋白酶的能力。【结论】杜比亚蟑螂肠道多种细菌具有产消化酶帮助降解大分子营养物质的功能,可通过协助食物消化影响宿主健康。菌株D12、D7和D11分别具有最强产纤维素酶、蛋白酶和脂肪酶的能力,是可进一步开发利用的肠道功能菌株资源。     

Mode of Association,Enzyme Producing Ability and Identification of Autochthonous Bacteria in the Gastrointestinal Tract of Two Indian Air-Breathing Fish,Murrel (<Emphasis Type="Italic">Channa punctatus</Emphasis>) and Stinging Catfish (<Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>)

The mode of association of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish species, the murrel, Channa punctatus and the stinging catfish, Heteropneustes fossilis was demonstrated through scanning and transmission electron microscopy (SEM and TEM). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. The TEM investigation indicated endocytosis and translocation of bacteria in the microvilli. The isolated bacterial strains (two each from the PI and DI of murrel and stinging catfish) were quantitatively evaluated for their extracellular amylase, cellulase and protease production. All the bacterial strains exhibited high cellulolytic activity than that of amylolytic and proteolytic enzymes. Only two strains, CPF1 and CPF2, isolated from the PI of murrel exhibited high proteolytic activity. Maximum amylase activity was exhibited by the strain, HFH5, isolated from the DI of stinging catfish. Totally six most promising enzyme-producing autochthonous bacterial strains were identified based on partial 16S rRNA gene sequence analytical results. All the strains showed close (92–99 %) similarity to Bacillus licheniformis.     

Xylanases of anaerobic fungus <Emphasis Type="Italic">Anaeromyces mucronatus</Emphasis>

The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase, xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase, laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa.