Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of Sucrose

A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutation from Lactobacillus amylovorus JCM 1126, the type strain defective in sucrose utilization. Exogenous sucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In a medium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed high invertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose and fructooligosaccharide were an identical β-fructofuranosidase, as judged from properties of partially purified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation of sucrose and not derepressed for the synthesis of invertase. Received: 23 May 2000 / Accepted: 26 June 2000     

Activity of salivary glands in secreting honey‐elaborating enzymes in two subspecies of honeybee (Apis mellifera L)

The activity of invertase, glucose oxidase and amylase in the cephalic (post‐cerebral) and thoracic salivary glands is determined in Egyptian and Carniolan honeybees (Apis mellifera L). For this purpose, three ages of worker bees are selected for enzyme assays. The results show that the three target enzymes are detected in the two glands during the three worker ages, except invertase, which cannot be detected in the cephalic gland of newly emerged bees of both subspecies. In both glands, the secretion of invertase is highest, followed by amylase and then glucose oxidase. In Carniolan bees, invertase secretion of the cephalic and thoracic glands increases gradually with age. In Egyptian bees, invertase increases with age only in the cephalic gland, whereas, in the thoracic gland, the highest secretion activity is detected in 10–15‐day‐old bees. The highest amounts of glucose oxidase and amylase in the cephalic gland are detected in newly emerged individuals of both Egyptian and Carniolan bees. In the thoracic gland, however, the highest activity of both enzymes is recorded only in newly emerged Egyptian bees. The results are discussed in the light of bee management and biological aspects of the two subspecies.     

Formations of Extracellular Isoamylase and Intracellular α-Glucosidase and Amylase(s) by Pseudomonas SB15 and a Mutant Strain

Pseudomonas SB15, which produces extracellular isoamylase, was found to produce intracellular alpha-glucosidase and amylase(s) when grown on maltose. A mutant strain (MS1) derived from it, which formed isoamylase constitutively, also produced these intracellular enzymes constitutively. The activities of the enzymes produced in the mutant strain were much greater than those induced in the parent strain.     

Effects of metsulphuron-methyl on amylase, invertase and xylanase activities in two soil types

Effects of metsulphuron-methyl on the activities of amylase, invertase and xylanase in loamy sand and clay were evaluated for up to 28 days under laboratory conditions. Metsulphuron-methyl at 1.0 microg/g caused a significant reduction in amylase, invertase and xylanase activities for the entire period of study, especially at 28 days incubation in both soils. The lowest activities of the three enzymes were observed in the presence of 5.0 microg/g at 28 days incubation.     

Temperature-sensitive forms of large and small invertase in a mutant derived from a SUC1 strain of Saccharomyces cerevisiae.

Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.     

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.     

Effect of NaCI Salinity on the Activities of Amylase and Invertase in Zea mays L. Pollen

Pollen collected from maize plants raised under 0, 80, 120 and160 mequiv 1^–1 salinity were used to determine the activitiesof amylase and invertase after 0 and 45 min of incubation inthe liquid basal germination medium. Amylase activity was higherin the ungerminated pollen collected from 120 and 160 mequiv1^–1 salinity while those pollen from lower salinity didnot show detectable amylase activity. However, 45 min afterincubation, the trend was reversed. Pollen collected from plantsraised under saline conditions showed increased invertase activitywhich further increased after 45 min of incubation in the basalgermination medium. The significance of changes in the activitiesof these hydrolytic enzymes in relation to pollen tube growthis discussed. Zea mays, salinity, pollen, amylase, invertase     

Activities of hydrolytic enzymes in callus cultures of tobacco during organogenesis

Starch, total sugars, reducing sugars and protein contents and the specific activities of hydrolytic enzymes such as amylase, Phosphorylase, soluble acid invertase, wall-bound acid invertase, sucrose synthetase, acid and alkaline phosphatases and ribonuclease were determined in root forming, shoot forming and non-organ-forming callus cultures of tobacco. Organ-forming cultures not only showed higher amounts of the above metabolites but also higher enzyme activities compared to non-organ-forming cultures. The activities of these enzymes in relation to organogenesis is discussed.     

‘台农1号’芒果果实发育过程中的糖分积累与相关酶活性研究

以‘台农1号’芒果为材料,测定了果实生长发育过程中淀粉、蔗糖、葡萄糖和果糖含量以及淀粉酶、蔗糖代谢相关酶———酸性转化酶(AI)、中性转化酶(NI)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)的活性,并对果实中糖组分与酶活性的关系进行了分析.结果显示,(1)台农1号芒果果实属于单S型生长曲线,发育前期主要积累淀粉、葡萄糖和果糖,果实成熟软化时,淀粉酶活性降至最低,淀粉水解,蔗糖快速积累.(2)酸性转化酶活性在果实整个发育过程中维持最高,完熟时略有降低;蔗糖磷酸合成酶在果实发育前期略有降低,完熟时升至最高;蔗糖合成酶和中性转化酶活性在整个发育期一直很低且较稳定.(3)淀粉含量与淀粉酶活性呈显著正相关,与SPS活性呈极显著负相关,蔗糖、葡萄糖含量均与SPS、SS呈显著、极显著的正相关;果糖含量与SS呈极显著的正相关.研究表明,芒果成熟时淀粉分解、酸性转化酶活性的降低,且蔗糖合成酶和蔗糖磷酸合成酶活性的增加是引起果实蔗糖积累的主要因子.     

Permeability of artificial lipid membranes to some enzymes

The permeability of artificial lipid membranes for six enzymes, e.g. RNAse, trypsin, amylase, aldolase, invertase and alkaline phosphatase, was studied. The permeability coefficient values for these enzymes were calculated. It was shown that the penetration process consists of several steps: adsorption of enzyme on the membrane surface, diffusion of enzyme molecules through the lipid layer and enzyme desorption into the surrounding solution. The results obtained suggest that the diffusion of the enzyme molecules through the lipid layer is the limiting step of the penetration process.     

Characteristics of the Kabicidin Resistant Mutant of Fusarium sp. Having a High Productivity of Alkaline Protease

In order to elucidate the biochemical mechanism of the alkaline protease accumulation from n-paraffins by a kabicidin-resistant mutant of Fusarium sp., the cell constituents and the extracellular products of the mutant strain were compared with those of the parent strain. No prominent differences in the cell constituents were observed between the parent and the mutant. From the analysis of the extracellular products, however the mutant was found to have a high productivity of some hydrolytic enzymes, such as amylase and ribonuclease, and ergosterol which is a structural constituent of fungal cell membrane. The relationship of secretion of ergosterol, resistance to kabicidin and accumulation of alkaline protease is discussed.     

Temperature-sensitive Saccharomyces cerevisiae mutant defective in lipid biosynthesis.

A temperature-sensitive mutant of Saccharomyces cerevisiae (DAM303) is described that exhibits an early defect in lipid biosynthesis at the restrictive growth temperature, 37 degrees C. This strain rapidly lost viability after 1 h of incubation at 37 degrees C, and this was accompanied by a significantly reduced incorporation of 32Pi into cellular lipid and an accumulation of [1-14C]acetate into the free fatty acid fraction. The temperature-sensitive DAM303 mutation failed to complement the sec13 mutation described by Novick et al. (Cell 21:205-215, 1980), and from analysis of invertase secretion in the temperature-sensitive DAM303 strain, it is clear that the loss of invertase secretion in the mutant occurs after the loss of phospholipid synthesis. Although the precise nature of the temperature-sensitive lesion in the DAM303 strain has still to be identified, the results from the study of this mutant indicate that a defect in lipid biosynthesis can be correlated with subsequent alterations in extracellular protein secretion and loss of other macromolecular functions including DNA, RNA, and protein syntheses. From studies of this mutant, two procedures of enriching for other temperature-sensitive mutants with defects in lipid biosynthesis have emerged: inositol overproduction and screening for increased buoyant densities.     

Effects of Salt-Alkaline Stress on Carbohydrate Metabolism in Rice Seedlings

The aim of this study was to investigate carbohydrate metabolism in rice seedlings subjected to salt-alkaline stress. Two relatively salt-alkaline tolerant (Changbai 9) and sensitive (Jinongda 138) rice cultivars, grown hydroponically, were subjected to salt-alkaline stress via 50 mM of salt-alkaline solution. The carbohydrate content and the activities of metabolism-related enzymes in the leaves and roots were investigated. The results showed that the contents of sucrose, fructose, and glucose in the leaves and roots increased under salt-alkaline stress. Starch content increased in the leaves but decreased in the roots under salt-alkaline stress. The activities of sucrose-phosphate synthase, sucrose synthase, amylase, and ADP-glucose pyrophosphorylase increased whereas the activities of neutral invertase and acid invertase decreased in the leaves under salt-alkaline stress. The activities of sucrose-phosphate synthase, sucrose synthase, amylase, neutral invertase, and acid invertase increased in the roots under salt-alkaline stress. In conclusion, salt-alkaline stress caused the accumulation of photosynthetic assimilates in the leaves and decreased assimilation export to the roots.     

Gene-Enzyme Relationships in Neurospora Invertase

A spontaneous, single-gene mutation responsible for a total lack of invertase activity in Neurospora crassa is described. The mutation is believed to lie in the structural gene for invertase, since an immunologically cross-reacting protein is made by the mutant strain. In addition, there was no evidence for a defect in regulation of invertase activity or synthesis by the following criteria. (i) The invertaseless condition was recessive in heterokaryons; (ii) no invertase inhibitor was found in mutant extracts by mixing experiments; and (iii) none of the several sugars able to induce activity in wild-type strains was able to induce activity in the mutant strain. It was also discovered that most of the wild-type enzyme (55 to 75%) cannot be washed free from the rapidly sedimenting cell debris. This finding provided additional support for the hypothesis that Neurospora invertase is located within or about the cell wall.     

氮沉降对杉木人工林土壤有机碳矿化和土壤酶活性的影响

为探讨氮沉降对亚热带森林土壤有机碳矿化及土壤酶活性的影响规律,在杉木人工林中开展了野外模拟N沉降试验。试验设计为4种处理,分别为N0（对照）、N1（60 kg N?hm-2?a-1）、N2（120 kg N?hm-2?a-1）和N3（240 kg N?hm-2?a-1）,每处理重复3次。通过28 d的培养后发现,各土层有机碳日均矿化量随培养时间的延长呈下降趋势,而有机碳累计矿化量则逐步增加。不同氮沉降处理下各土层有机碳累计矿化量总体趋势表现为：随着氮沉降量的增加而降低,日均矿化量降低幅度以N1最大,其次是N0和N2,N3降幅最小。相同N沉降处理下,参与土壤碳循环的6种主要酶（蔗糖酶、纤维素酶、淀粉酶、β-葡糖苷酶、多酚氧化酶、过氧化物酶）活性、土壤有机碳日均矿化量和有机碳累计矿化量均随土层加深而降低。氮沉降对6种土壤酶活性的影响存在差异,对纤维素酶和多酚氧化酶具有促进作用,而对淀粉酶和过氧化物酶表现出一定的抑制作用;中-低氮沉降（N1、N2）对蔗糖酶无影响,而对β-葡糖苷酶具有促进作用,高氮沉降（N3）促进了蔗糖酶活性,但抑制了β-葡糖苷酶活性。表层土壤中,土壤有机碳累积矿化量与土壤纤维素酶、β-葡糖苷酶、过氧化物酶活性呈显著正相关。因此,氮沉降促进了表层土壤纤维素酶、多酚氧化酶和蔗糖酶的活性,但在一定程度上抑制了淀粉酶和过氧化物酶,对土壤有机碳矿化也表现出明显的抑制作用。     

Biochemical studies of amylases in the silkworm, Bombyx mori L.: Comparative analysis in diapausing and nondiapausing strains

Different amylase enzymes were identified by analysis of digestive fluid and haemolymph in diapausing and nondiapausing strains of silkworm, Bombyx mori. The diapausing strain showed negligible digestive amylase activity at a pH range of 3–11, while the nondiapausing strain registered strikingly higher amylase activity at pH 9.2. Higher levels of undigested starch was found in the faecal matter of the diapausing strain, which is consistent with the negligible digestive amylase activity. Development specific expression of haemolymph amylase activity was seen in nondiapausing and diapausing strains. In the nondiapausing strain the digestive amylase activity was at its peak during intermoult and depressed during moult. PAGE analysis revealed the occurrence of only anodal digestive and haemolymph amylases in the diapausing strain, whereas both cathodal and anodal enzymes were seen in the digestive fluid and haemolymph of the nondiapausing strain.     

Transfer and expression of mutations in pleiotropic genes determining the synthesis of extracellular enzymes in Bacillus subtilis

The mutations in the genes determining the levels of extracellular enzymes production were transferred into Bacillus subtilis 168 strain by genetic transformation technique. The method used has permitted registering the transfer of pleiotropic genes. Seven amylase producers were detected among 126 his+ transformants screened, as well as five metalprotease producers among 246 gly+ transformants. Cotransfer of pap and hpr mutant genes linked with his+ or gly+ genes might result in finding the producers among the mentioned prototrophic transformants. Selection of linked markers in transformation, presented in the paper, is discussed to be a useful technique for obtaining producer strains for industrial production.     

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.     

Biochemical characteristics of new thermosensitive secretory mutants of yeasts

New thermosensitive mutants of the yeast Saccharomyces cerevisiae which block the secretion of periplasmic enzymes at restriction temperature have been obtained. These mutants accumulate active low molecular weight and mature invertase species in the cell; the buoyant density of the cells in a Percoll gradient is higher than that in the wild strain cells. The mutant cells transferred to permissive temperature (25 degrees C) in the absence of protein synthesis can secrete some amount of accumulated invertase. It was found that the secretory defects of conditional mutants do not affect the activity of cytoplasmic enzymes (e.g., alcohol dehydrogenase) or the level of total protein synthesis and glycosylation and do not induce non-specific disturbances in energy metabolism and plasma membrane functions at restriction temperature. Some strains of new secretory mutants revealed uncoupled defective secretion of periplasmic enzymes and intrinsic membrane proteins (proline permease). The possibility of branching of the secretory pathway for periplasmic enzymes and cytoplasmic membrane proteins is discussed.