Biosynthesis of Hydrolytic Enzymes during Cocultivation of Macro- and Micromycetes

Effects of cocultivation of higher basidiomycetes and zygomycetes on biosynthesis of cellulases, amylases, and proteases were studied. Four optimal pairs of cultures of fungi were selected. Of these, three pairs belonged to higher fungi and one pair was constituted by fungi of distinct ecological groups, a macromycete and a micromycete. The activities of amylase and protease were 1.5–2 times higher and the activity of cellulase was lower during the growth of associations of higher fungi. The mixed association of the macromycete Schizophyllum commune and the micromycete Mucor sp. was the most active producer of hydrolytic enzymes. During the growth of this mixed association, increases of 4 and 1.5 times were observed in the activity of endoglucanase and protease, respectively, paralleled by stimulation of amylase formation.     

Exocellular enzyme activity of dermatophytes and other fungi isolated from ruminants in Southern Iraq

Sixteen fungal species were isolated from 182 specimens collected from four ruminants (buffalo, camel, cattle and sheep) in Southern Iraq. Fungi represented by five species of dermatophytes and eleven species of other fungi were screened for the activity of four enzymes; keratinase, proteinase, lipase and amylase. Keratinase was found to be produced by all of the dermatophytes and non-dermatophytes, except for Paecillomyces variottii and Scytalidium lignicola. However, high keratinase activity was expressed by the dermatophytic species particularly by Trichophyton mentagrophytes var. erinacei and Microsporum gypseum. Three dermatophytes viz. M. gypseum, T. verrucosum and T. mentagrophytes var. nodulare were capable of producing protease, lipase and amylase. Although, T. mentagrophytes var. erinacei showed high protease activity, it did not produce lipase and amylase. On the contrary most of the non-dermatophytic species revealed protease and lipase activities higher than the dermatophytes. The Curvularia spp. isolates showed the highest protease and amylase activity, while Aspergillus parasiticus revealed the highest activity of lipase and amylase. No correlation was observed between enzyme activity and the growth rate of the examined fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

中华真地鳖中肠主要消化酶的活性研究

以中华真地鳖EupolyphagasinensisWalker为研究材料,测定人工饲养和野生地鳖虫在不同生长阶段消化酶的活性以及温度及pH对人工饲养地鳖虫中肠消化酶活性的影响。结果表明,在地鳖虫生长发育过程中,蛋白酶和脂肪酶活性随发育而逐渐增强,淀粉酶活性却随发育而逐渐减弱。在低龄若虫、高龄若虫和成虫阶段,人工饲养地鳖虫蛋白酶活力比野生地鳖虫低,人工饲养地鳖虫淀粉酶和脂肪酶活力比野生地鳖虫高;在30～60℃的范围内,人工饲养地鳖虫蛋白酶、淀粉酶的适宜温度范围为40～50℃,脂肪酶的适宜温度范围为35～45℃;蛋白酶、淀粉酶和脂肪酶的适宜pH范围分别为6.5～7.5,5.6～6.4和7.5～8.5。     

Effect of iso-osmotic levels of salts and PEG-6000 on enzymes in germinating pea seeds

Effects of iso-osmotic levels of salts (NaCl, CaCl₂, Na₂SO₄) and PEG-6000 on the activity of hydrolytic and nitrogen assimilatory enzymes in pea embryo axis and coty ledon were studied. The activity of nitrate reductase and nitrite reductase in embryo axis and cotyledon and the activity of protease and α-amylase in cotyledon decreased with decreasing medium osmotic potential as compared to control at all the stages of seedling growth. The activity of protease and amylase increases with increasing levels of stress in embryo axis. Sodium chloride induced, stress had more deleterious effects on the activity of nitrate reductase, nitrite reductase and αamylase followed by other salts and PEG-6000. On the other hand, CaCl₂ induced salt stress was more depressive for protease activity. The maximum increase in the activity of protease and amylase was observed in embryo axis at higher concentration of salts and PEG-6000.     

Correlation between the fatty acid composition and the activity of extracellular enzymes from Bacillus caldolyticus

Summary Bacillus caldolyticus, grown at 70°C, produces a highly active extracellular amylase and protease. Both enzymes are formed either within the membrane, or at its inner surface. The activity of both extracellular enzymes was found to decline drastically when brain-heart infusion was omitted from the medium. A simultaneous increase of both enzymes inside the cell was observed. The shifting in extra- and intra-cellular activity was caused by changes in membrane composition due to the increase of anteiso-odd and n-even, and the decrease of iso-odd fatty acids. Membrane composition and enzymic activity could be influenced by the addition of either leucine or iso-leucine as precursors for the synthesis of branched-chain fatty acids: In presence of leucine the anteiso-odd and n-even fatty acids returned to their normal level, while the iso-odd fatty acids increased. Simultaneously the extracellular protease activity increased, and the intracellular activity declined. Growth in amylose-medium supplied with leucine lead to a decrease of both the intra- and extracellular amylase, and changes in the fatty acid composition of the membrane which could not be restored by transfer of the organism to complete media. Addition of iso-leucine first lead to a sharp decrease of extracellular protease and a drastic increase of intracellular protease activity, accompanied by an increase of anteiso-odd and n-even fatty acids, and a decrease of iso-odd compounds. After the second growth in presence of iso-leucine the intra- and extra-cellular protease activity was reversed, and thus showed a return to the starting situation. The reversal is accompanied by the preferential incorporation of fatty acids with a higher melting point into the membrane. Extracellular amylase activity was found to increase after the first growth with iso-leucine, and to decline sharply after the second culture with iso-leucine, together with a very high intracellular amylase activity at that point. Extra- and intra-cellular amylase activity both declined upon growth in complete medium, while the fatty acid distribution remained different from the initial composition.     

Effect of two systemic fungicides on cellulose decomposing fungi of tomato plants and on some enzymatic activities

Kocide 101 (77% cupric hydroxide) and Ridomil plus (15% metalaxyl and 35% copper oxychloride) were used in the treatment of tomato plants. The two fungicides exerted a depressive effect on the total counts and on the individual cellulose decomposing fungal species associated with the roots and shoots of tomato. When these fungicides were incorporated in the liquid culture medium specified for growth and extracellular enzyme production by some selected fungal species, there was a significant reduction in mycelial growth as well as in amylase, cellulase, lipase and protease production by the fungi tested, particularly at the higher doses (200-400 ppm). Exceptions were observed with lower doses (50 and 100 ppm) especially in case of Aspergillus flavus, Cunninghamella echinulata, Penicillium chrysogenum and Fusarium oxysporum grown for amylase or cellulase production.     

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

The synergistic ability of some wood-degrading fungi to transform lignins and lignosulfonates on various media

Summary In order to investigate the ligninolytic activity with mixed cultures of wood-degrading fungi, and the influence of various growth conditions on this activity, 50 wood-degrading fungi were tested for ligninolysis in pure culture and in pair-wise combinations according to a simple plate test recently developed in this laboratory. It was found that a synergistic degradation of lignin and of lignosulfonate was common among fungi inoculated pair-wise on lignin or lignosulfonate media; decomposition was enhanced in the zone where the two mycelia interacted. This synergistic effect was noted with pairs of two different white-rot fungi, with pairs of one white-rot and one brown-rot fungus, and with pairs of one white-rot and one soil Deuteromycete.Lignosulfonate was more susceptible to the synergistic action of pairs of fungi than was lignin. The synergistic attack on lignosulfonate was more pronounced on a meager medium than on a carbohydrate-rich one. On the contrary, the ligninolysis with pure cultures of the fungi was more pronounced on the carbohydrate-rich medium, and lignin was decomposed more easily than was lignosulfonate.     

Relationship between physiological measurements (SFG -scope for growth-) and the functionality of the digestive gland in Mytilus galloprovincialis

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed.     

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved in pathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation, proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), hence indicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity of protease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase- 2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed that highest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidic pH 3. Maximum amylolytic activity of V. lecanii on the 7^th day of inoculation was at pH 3 i.e. in an acidic environment in contrast to neutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi is specific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence a significant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.     

不同生态环境中铜绿丽金龟幼虫肠道细菌 产消化酶活性比较

目的测定不同生态环境中铜绿丽金龟幼虫肠道细菌产消化酶活性。方法采用平板透明圈法和分光光度计法。结果平板透明圈法测得废弃菜园和花生田中铜绿丽金龟幼虫肠道细菌产蛋白酶和淀粉酶活性差异无统计学意义(F=1.089、0.4963,P0.05),而细菌的产纤维素酶活性有显著差异,其中花生田铜绿丽金龟幼虫肠道中分离到的粘质沙雷菌产酶活性显著高于其他菌株;分光光度计测得的不同生态环境中铜绿丽金龟幼虫肠道细菌的产蛋白酶、淀粉酶和纤维素酶活性差异均有统计学意义(F=461.565、42.349、18.7673,P0.05)。从变异系数来看,分光光度计法测得的蛋白酶、淀粉酶和纤维素酶的变异程度较低,而脂肪酶测定时平板透明圈法的变异系数较低。结论平板透明圈法和分光光度计法均可用于铜绿丽金龟幼虫肠道细菌产消化酶活性测定。通过分析研究比较,分光光度计法适于测定细菌的产蛋白酶、淀粉酶和纤维素酶活性,平板透明圈法适于测定细菌的产脂肪酶活性。     

Application of Bacillus-antagonists for biocontrol of fungi degrading raw wood

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.     

云斑尖塘鳢消化酶活力的研究

本文采用酶学分析方法研究了云斑尖塘鳢在正常摄食状态与饥饿的状态下胃、肠及肝胰脏组织中蛋白酶、淀粉酶和脂肪酶的活性。结果显示,在30℃的条件下,正常摄食组样本在酸性条件下的蛋白酶活力表现为:胃后肠肝胰脏前肠,中性和碱性条件下:后肠肝胰脏前肠及胃;饥饿组样本仅有胃表现出较高的酸性蛋白酶活性,其他器官的蛋白酶活性均很低。在正常和饥饿实验组中肝胰脏的淀粉酶活性均高于其他器官,胃肠的淀粉酶活性均较低。正常摄食组中脂肪酶活力后肠肝胰脏;而在饥饿组中仅有肝胰脏检测到脂肪酶活性。结果表明,云斑尖塘鳢适度饥饿组较正常摄食组消化酶活性大幅降低;其高蛋白酶活力及中等脂肪酶活力与其肉食性相一致;此外云斑尖塘鳢也具备少量的淀粉消化能力。     

盐度对花鳗鲡和太平洋双色鳗鲡幼鳗生长性能及消化酶活力的影响

文章研究了不同盐度对花鳗鲡(Anguilla marmorata)幼鳗和太平洋双色鳗鲡(A. bicolor pacifica)幼鳗生长性能及消化酶活力的影响。将花鳗鲡幼鳗(9.760.36) g和太平洋双色鳗鲡幼鳗(11.820.04) g分别在淡水(盐度0)与盐度5、10、18水体中养殖30d, 测量每组实验鱼总重后检测胃、肠道和肝脏蛋白酶、淀粉酶和脂肪酶的活力。结果表明, 花鳗鲡和太平洋双色鳗鲡在各盐度处理中存活率均为100%, 未出现死亡。两种鳗鲡在淡水中生长良好, 特定生长率最高, 而饵料系数最低。盐度对花鳗鲡幼鳗和太平洋双色鳗鲡幼鳗消化酶活力的影响存在差异, 其中花鳗鲡胃、肠道和肝脏蛋白酶活力在各盐度处理中均无显著变化(P0.05), 淀粉酶和脂肪酶活力均随盐度的增加而下降; 太平洋双色鳗鲡胃蛋白酶活力在盐度10时最大, 肝蛋白酶活力在盐度18时最大, 而淀粉酶和脂肪酶活力在各盐度处理组无显著变化(P0.05)。这表明盐度对花鳗鲡胃、肠道和肝脏的淀粉酶和脂肪酶活力具有抑制作用, 对太平洋双色鳗鲡的蛋白酶活力有一定的激活作用。在相同盐度条件下, 不同消化器官中同种消化酶活力存在差异, 各盐度的两种鳗鲡肠道中淀粉酶和脂肪酶的活力均显著高于肝脏和胃(P0.05), 胃中蛋白酶活力高于肝脏和肠道, 但不显著(P0.05)。研究发现两种鳗鲡体内脂肪酶活力相对较高, 表明其对脂肪具有较强的消化能力。建议在配制花鳗鲡幼鳗和太平洋双色鳗鲡幼鳗饲料时, 适当提高粗脂肪比例, 有助于促进对营养物质的消化吸收, 提高养殖效益。       

Extracellular enzyme production by haploids,heterocaryons and diploids of Aspergillus nidulans

Summary Extracellular amylase, lipase and protease produced by haploids, diploids and heterocaryons of Aspergillus nidulans were analysed. Three morphologically normal strains and 8 morphologic mutants as well as various genetic combinations of the 11 strains were examined in solid culture medium containing specific substrates. The enzyme production of each strain was determined by measuring the halo around the colony. It was observed that the colonies showing less growth also showed more alterations in enzyme production. The compact strains (BVIII and B6) and the slow-growing heterocaryons (pp+M32 and pp+M35) showed the highest enzymatic index for the three enzymes simultaneously. If colony growth is not considered, then for amylase and protease the highest values were reached by some diploid and heterocaryons and for lipase by one morphological strain. The results showed that morphological mutants and some combinations could be used for higher production of amylase, lipase and protease.     

意大利蜜蜂工蜂唾腺和中肠消化酶活性比较研究

对意大利蜜蜂 Apis mellifera ligustica L工蜂唾腺和中肠中淀粉酶、蛋白酶、蔗糖酶、果胶酶和海藻糖酶5种消化酶进行活性测定,发现巾肠的蛋门酶和蔗糖酶活性较唾腺高,有显著性差异;唾腺的果胶酶活性较中肠高,有显著性差异;唾腺与中肠的淀粉酶和海藻糖酶没有显著性差异.表明意大利蜜蜂工蜂对蔗糖和蛋白的消化主要由中肠分泌的消化酶来完成,唾腺起辅助作用;对果胶的消化主要在唾腺,中肠起辅助作用;而在淀粉和海藻糖的消化过程中唾腺和中肠都有很重要的作用.     

怀头鲇早期发育阶段消化酶及碱性磷酸酶活性变化(英文)

采用动物性饵料和人工饲料培育1～10日龄怀头鲇(Silurus soldatovi)仔稚鱼,分析测定了全鱼酸性、碱性蛋白酶、淀粉酶、脂肪酶以及碱性磷酸酶的活性。结果表明:孵化后3天开口期仔鱼已具有较高的碱性蛋白酶活性,5日龄时碱性蛋白酶比活力达到较高值,8日龄时出现低值,总体变化呈波动上升趋势;酸性蛋白酶活性在1～8日龄处于较低水平,8日龄后开始迅速升高;淀粉酶活性在5日龄左右达到最高值,随后酶活性开始下降至较低水平;脂肪酶活性变化波动较大,表现为双峰型,两个峰值分别出现在3～4日龄和6～8日龄。摄食动物性饵料仔稚鱼消化酶活性和碱性磷酸酶活性均高于摄食人工饲料。在整个早期发育过程中,碱性蛋白酶比酸性蛋白酶活性高,碱性蛋白酶、淀粉酶比活力在约8日龄仔稚鱼转变期明显下降,而酸性蛋白酶活性开始迅速升高,这说明消化酶活性的变化与仔稚鱼发育过程中消化机能转换具有相关性。怀头鲇在10日龄内碱性磷酸酶活性呈上升趋势,表明怀头鲇胃肠道功能的逐步发育完善。     

Effect of amino compounds on alkaline amylase production by alkalophilic Bacillus sp.

Of the amino compounds investigated, β-alanine, dl-norvaline and d-methionine were effective for the production of alkaline amylase by alkalophilic Bacillus no. A-40-2. The addition of 0.5% dl-norvaline and 0.5% d-methionine to the culture medium increased amylase production 1.7-fold, while they repressed growth slightly or strongly. There was no relation between amylase yield and the extracellular protein amount. Because alkaline protease activity was negligible in the culture fluid, these compounds might change the regulation of amylase synthesis and/or make amylase excretion easier.     

高效降解蛋白枯草芽孢杆菌的筛选及促建鲤生长的研究

【目的】从建鲤肠道分离并优选出了一株对蛋白具有强效降解力的菌株,探究其临床生产效果。【方法】使用牛奶平板进行筛选,经常规细菌学鉴定和16S rDNA序列分析确定A7菌株为枯草芽孢杆菌,通过临床饲喂试验探究其对建鲤促生长作用。【结果】A7菌株在牛奶平板上的水解圈直径可达27.5 mm,其最佳固体发酵条件为温度28°C、接种量5%(1.2×109 CFU/mL)、料水比1.0:1.2和发酵时间72 h。临床饲喂试验结果表明,饵料中添加0.5%、1.0%和1.5%的A7株菌粉均能促建鲤生长。其中,添加量为1.0%的试验组,鱼体增重率和蛋白利用率最高,饵料系数最低,与产品对照组和空白对照组相比,均达到了显著性差异(P0.05)。此外,随A7株菌粉添菌量的增加,各试验组肝胰脏和肠道的蛋白酶及淀粉酶活力出现先增大后减小趋势,与产品对照组和空白对照组相比,添加1.0%的试验组增幅最大(P0.05)。研究发现,试验组鱼肌肉中粗蛋白和粗脂肪含量较空白对照组也有明显的增减趋势(P0.05)。【结论】饵料中添加A7株菌粉,可有效促进鱼的生长,降低饵料系数,提高肝胰脏和肠道的蛋白酶及淀粉酶活力,并提高鱼肌肉中的蛋白含量和降低脂肪含量。