Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.     

Mobilization and utilization of cyanogenic glycosides: the linustatin pathway

In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside linamarin (2-β-d-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the endosperm. After onset of germination, the cyanogenic diglucoside linustatin (2-[6-β-d-glucosyl-β-d-glucopyranosyloxy]-2- methylpropionitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The linustatin-splitting diglucosidase and the β-cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at this time. Based on these observations the following pathway for the in vivo mobilization and metabolism of cyanogenic glucosides is proposed: storage of monoglucosides (in the endosperm)—glucosylation—transport of the diglucoside (out of the endosperm into the seedling)—cleavage by diglucosidase—reassimilation of HCN to noncyanogenic compounds. The presence of this pathway demonstrates that cyanogenic glucosides, typical secondary plant products serve in the metabolism of developing plants as N-storage compounds and do not exclusively exhibit protective functions due to their repellent effect.     

Purification, characterization, and localization of linamarase in cassava

We have purified cassava (Manihot esculenta) linamarase to apparent homogeneity using a simplified extraction procedure using low pH phosphate buffer. Three isozymes of cassava linamarase were identified in leaves based on differences in isoelectric point. The enzyme is capable of hydrolyzing a number of β-glycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 55°C. Immunogold labeling studies indicate that linamarase is localized in the cell walls of cassava leaf tissue. Since linamarin must cross the cell wall following synthesis in the leaf for transport to the root, it is likely that linamarin must cross the cell wall in a nonhydrolyzable form, possibly as the diglucoside, linustatin. In addition, we have quantified the levels of linamarin and linamarase activity in leaves of cassava varieties which differ in the linamarin content of their roots. We observed no substantial differences in the steady state linamarin content or linamarase activity of leaves from high or low (root) cyanogenic varieties. These results indicate that the steady state levels of linamarin and linamarase in leaves of high and low cyanogenic varieties are not correlated with the varietal differences in the steady state levels of linamarin in roots.     

Compartmentation of cyanogenic glucosides and their degrading enzymes

Whereas high activities of β-glucosidase occur in homogenates of leaves of Hevea brasiliensis Muell.-Arg., this enzyme, which is capable of splitting the cyanogenic monoglucoside linamarin (linamarase), is not present in intact protoplasts prepared from the corresponding leaves. Thus, in leaves of H. brasiliensis the entire linamarase is located in the apoplasmic space. By analyzing the vacuoles obtained from leaf protoplasts isolated from mesophyll and epidermal layers of H. brasiliensis leaves, it was shown that the cyanogenic glucoside linamarin is localized exclusively in the central vacuole. Analyses of apoplasmic fluids from leaves of six other cyanogenic species showed that significant linamarase activity is present in the apoplasm of all plants tested. In contrast, no activity of any diglucosidase capable of hydrolyzing the cyanogenic diglucoside linustatin (linustatinase) could be detected in these apoplasmic fluids. As described earlier, any translocation of cyanogenic glucosides involves the interaction of monoglucosidic and diglucosidic cyanogens with the corresponding glycosidases (Selmar, 1993a, Planta 191, 191–199). Based on this, the data on the compartmentation of cyanogenic glucosides and their degrading enzymes in Hevea are discussed with respect to the complex metabolism and the transport of cyanogenic glucosides.     

Relation of glycosidases to bean hypocotyl growth

The enzymes β-glucosidase, α-glucosidase, β-galactosidase, α-galactosidase, and β-xylosidase were detected in Phaseolus vulgaris L. var. Red Kidney bean hypocotyl tissue throughout the first 13 days of development with p-nitrophenyl glycosides as substrates. Activities of all enzymes except β-glucosidase declined as a function of increasing tissue age. In contrast, β-glucosidase activity increased rapidly 3 days after imbibition to a maximal activity at 5 days and then subsided to one-third the maximum by day 7. This activity peak immediately preceded the logarithmic phase of hypocotyl growth. This enzyme is strongly associated with cell walls during extraction, suggesting that it is wall-bound in situ. Various polysaccharide substrates were used to evaluate the specificity of this enzyme.     

Transport of cyanogenic glucosides: linustatin uptake by Hevea cotyledons

The ¹⁴C-labelled cyanogenic glucosides linustatin (diglucoside of acetone cyanohydrin) and linamarin (monoglucoside of acetone cyanohydrin), prepared by feeding [¹⁴C]valine to plants of Linum usitatissimum L., were applied to cotyledons of Hevea brasiliensis Muell.-Arg. in order to study their transport. Both [¹⁴C]-linustatin and [¹⁴C]linamarin were efficiently taken up by the cotyledons. Whereas ¹⁴C was recovered completely when [¹⁴C]linustatin was applied to the seedling, only about one-half of the radioactivity fed as [¹⁴C]linamarin could be accounted for after incubation. This observation is in agreement with the finding that apoplasmic linamarase hydrolyzes linamarin but not the related diglucoside linustatin. These data prove that, in vivo, linamarin does not occur apoplasmically and that linustatin, which is exuded from the endosperm, is taken up by the cotyledons very efficiently. Thus, these findings confirm the linustatin pathway (Selmar et al. 1988, Plant Physiol. 86, 711–716), which describes mobilization and transport of the cyanogenic glucoside linamarin, initiated by the glucosylation of linamarin to yield linustatin. When linustatin is metabolized to non-cyanogenic compounds, in Hevea this cyanogenic diglucoside is hydrolyzed by a diglucosidase which splits off both glucose molecules simultaneously as one gentiobiose moiety (Selmar et al. 1988). In contrast, [¹⁴C]linustatin, which is taken up by the cotyledon, is not metabolized but is reconverted in high amounts to the monoglucosidic [¹⁴C]linamarin, which then is temporarily stored in the cotyledons. These data demonstrate that in Hevea, besides the simultaneous diglucosidase, there must be present a further diglucosidase which is able to hydrolyze cyanogenic diglucosides sequentially by splitting off only the terminal glucose moiety from linustatin to yield linamarin. From this, it is deduced that the metabolic fate of linustatin, which is transported into the source tissues, depends on the activities of the different diglucosidases. Whereas sequential cleavage — producing linamarin — is purely a part of the process of linamarin translocation (using linustatin as the transport vehicle), simultaneous cleavage, producing acetone cyanohydrin, is part of the process of linamarin metabolization in which the nitrogen from cyanogenic glucosides is used to synthesize non-cyanogenic compounds.     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

The leaf, inner bark and latex cyanide potential of Hevea brasiliensis: Evidence for involvement of cyanogenic glucosides in rubber yield

The latex of Hevea brasiliensis, expelled upon bark tapping, is the cytoplasm of anastomosed latex cells in the inner bark of the rubber tree. Latex regeneration between two tappings is one of the major limiting factors of rubber yield. Hevea species contain high amounts of cyanogenic glucosides from which cyanide is released when the plant is damaged providing an efficient defense mechanism against herbivores. In H. brasiliensis, the cyanogenic glucosides mainly consist of the monoglucoside linamarin (synthesized in the leaves), and its diglucoside transport-form, linustatin. Variations in leaf cyanide potential (CNp) were studied using various parameters. Results showed that the younger the leaf, the higher the CNp. Leaf CNp greatly decreased when leaves were directly exposed to sunlight. These results allowed us to determine the best leaf sampling conditions for the comparison of leaf CNp. Under these conditions, leaf CNp was found to vary from less than 25 mM to more than 60 mM. The rubber clones containing the highest leaf CNp were those with the highest yield potential. In mature virgin trees, the CNp of the trunk inner bark was shown to be proportional to leaf CNp and to decrease on tapping. However, the latex itself exhibited very low (if any) CNp, while harboring all the enzymes (β-d-diglucosidase, linamarase and β-cyanoalanine synthase) necessary to metabolize cyanogenic glucosides to generate non-cyanogenic compounds, such as asparagine. This suggests that in the rubber tree bark, cyanogenic glucosides may be a source of buffering nitrogen and glucose, thereby contributing to latex regeneration/production.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

Genetic Determination of the Constitutive Biosynthesis of Phospho-β-Glucosidase A in Escherichia coli K-12

Escherichia coli wild-type cells form constitutively the enzyme phospho-β-glucosidase A, which has a high affinity for phosphorylated aromatic β-glucosides and a low affinity for phosphorylated β-methyl-glucoside. Phospho-β-glucosidase B and β-glucoside permease I are formed in aromatic β-glucoside-fermenting mutants. Mutants lacking phospho-β-glucosidases A and B have been isolated. These mutants showed a reduced rate of inducibility of the β-glucoside permease I. The restoration of phospho-β-glucosidase A or B activity resulted in an increased rate of induction of the β-glucoside permease I. The presence of the phospho-β-glucosidases was not required for the constitutive biosynthesis of the β-glucoside permease. Mutants selected for growth on β-methyl-glucoside as carbon source showed an increased level of constitutive phospho-β-glucosidase A activity. Gene bglD, the structural gene for phospho-β-glucosidase A, was mapped between the pyrE locus and the cluster bgl loci, whereas bglE, the regulatory site determining the hyperproduction of phospho-β-glucosidase A, was mapped between the bgl and ilv clusters. The bglE locus appears to have a regulatory effect on the expression of the bglD gene.     

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Inhibition of glycosidases by aldonolactones of corresponding configuration: The C-4- and C-6-specificity of β-glucosidase and β-galactosidase

1. In barley, β-glucosidase and β-galactosidase are separate enzymes. The former also displays β-d-fucosidase activity. 2. In the limpet, Patella vulgata, β-glucosidase activity is associated with the β-d-fucosidase, previously shown to be a separate entity from the β-galactosidase also present. 3. Almond emulsin presents all three activities as a single enzyme. Each is equally inhibited by glucono-, galactono- and d-fucono-lactone. 4. In rat epididymis, there is no significant β-glucosidase activity, nor is there appreciable inhibition of the β-galactosidase and β-d-fucosidase activities of the preparation by gluconolactone.     

Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

Background

Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/Principal Findings

Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females'' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/Significance

Herein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.     

Partial Characterization and Subcellular Localization of Three alpha-Glucosidase Isoforms in Pea (Pisum sativum L.) Seedlings

Three isoforms of α-glucosidase (EC 3.2.1.20) have been extracted from pea (Pisum sativum L.) seedlings and separated by DEAE-cellulose and CM-Sepharose chromatography. Two α-glucosidase isoforms (αG1 and αG2) were most active under acid conditions, and appeared to be apoplastic. A neutral form (αG3) was most active near pH 7, and was identified as a chloroplastic enzyme. Together, the activity of αG1 and αG2 in apoplastic preparations accounted for 21% of the total acid α-glucosidase activity recovered from pea stems. The vast majority (86%) of the apoplastic acid α-glucosidase activity was due to αG1. The apparent K_m values for maltose of αG1 and αG2 were 0.3 and 1.3 millimolar, respectively. The apparent K_m for maltose of αG3 was 33 millimolar. The respective native molecular weights of αG1, αG2, and αG3 were 125,000, 150,000, and 110,000.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

Relationship of Cyanogenic Capacity (HCN-c) of the Rubber Tree Hevea brasiliensis to Susceptibility to Microcyclus ulei, the Agent Causing South American Leaf Blight

Liberation of HCN from cyanogenic plant tissue depends on cyanogen content (HCN-potential), cyanogen splitting enzymes, cyanohydrin cleaving activity (hydroxynitrile lyase) and nonenzymatic cyanide detoxifying compounds. The maximal amount of HCN potentially set free is governed by the total cyanogen content, whereas the velocity of HCN liberation depends on enzymatic activities (β-glucosidase [β-G], hydroxynitrile lyase [HN]). Plants revealing a high HCN-potential and a high β-glucosidase activity generally are susceptible to infection with Microcyclus ulei. Based on the data of HCN-p and β-G activity of different Hevea species a proposal for future screening work in Hevea resistance selection is given.     

Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

High Production of β-Glucosidase in Schizophyllum commune: Isolation of the Enzyme and Effect of the Culture Filtrate on Cellulose Hydrolysis

Optimization experiments with response surface statistical analysis were performed with Schizophyllum commune to obtain high β-glucosidase yields. The factors in the optimization experiment were the concentrations of cellulose, peptone, and KH₂PO₄. Their optimal values were 3.2, 3.0, and 0.2 g/100 ml, respectively. Enzyme assays revealed very high β-glucosidase (22.2 U/ml) and cellobiase (68.9 U/ml) yields. The avicelase yield was low as compared with that from Trichoderma reesei. Mixtures of S. commune and T. reesei culture filtrates caused faster and more extensive saccharification of Avicel than could be achieved by either filtrate alone. A β-glucosidase was isolated and purified from the optimized culture filtrate of S. commune. The electrophoretic mobility of the purified β-glucosidase indicated a molecular weight of 97,000. The amino acid composition was similar to that of β-glucosidase from T. reesei. The acidic (aspartate and glutamate) residues or their amides or both made up approximately 20% of the protein. The NH₂-terminal amino acid of the enzyme was histidine.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.