New Strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α-Amylase Active at Alkaline pH

Two species of Bacillus producing thermostable α-amylase with activity optima at alkaline pH are reported here. These organisms were isolated from soil and have been designated as Bacillus licheniformis CUMC 305 and B. coagulans CUMC 512. The enzymes released by these two species were partially purified up to about 81- and 72-fold respectively of the initial activity. The enzyme from B. licheniformis showed a wide temperature-range of activity, with optimum at 91°C. At this temperature it remained stable for 1 h. It retained 40–50% activity at 110°C and showed only 60% of its activity at 30°C. The enzyme showed a broad pH range of activity (4–10) retaining substantial activity on the alkaline side. The optimum pH was 9·5. The enzyme of B. coagulans showed activity up to 90°C, with optimum at 85°C and had a wide pH range with optimum at 7·5–8·5. The hydrolysis pattern of the substrate starch by these enzymes indicated that glucose, maltose, maltotriose and maltotetraose are the principal products rather than higher oligosaccharides.     

Proteolytic activity from a thermophilic Streptomyces megasporus strain SDP4

Multiple proteases secreted by a thermophilic actinomycete Streptomyces megasporus SDP4 after 18 h of growth at 55 °C are reported. The enzyme preparation exhibited activity over a broad pH and temperature range of pH 6–12 and 25–85 °C, respectively. Optimum activity was observed at pH 8·0, pH 10·0 and 55 °C and was calcium independent. Thermostability was enhanced in the presence of 0·01 mol l⁻¹ calcium ions and half-life was 30 min at 85 °C. The enzyme was active in the presence of SDS. Both, EDTA and PMSF were partially inhibitory, indicating the presence of serine and metal requiring proteases. Three active zones in the range of 90–30 kDa were detected post-electrophoretically.     

Keratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus strain SD8

Keratinolytic activity was detected in the culture broth of feather-degrading thermophilic Streptomyces thermoviolaceus SD8. The crude enzyme was concentrated by precipitation with 80% saturation of ammonium sulphate and desalted by SephadexG-10–120 gel chromatography followed by lyophilization. The specific activity of the enzyme was enhanced 50-fold. PAGE analysis indicated a monomeric form with a molecular weight of 40 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 °C, respectively. The enzyme was stable at a pH range of 6·5–8·5 and up to 65 °C. The enzyme could hydrolyse fibrin, muscle, collagen, nail and hair and could produce leucine, threonine and tyrosine from feather. It could be a useful enzyme for waste treatment by promoting hydrolysis of the above substances in the sewage, or it could be used for animal feed preparation.     

Purification and characterization of glucoamylase of Aspergillus terreus NA-170 mutant

A catabolite-derepressed mutant strain of Aspergillus terreus NA-170 was isolated by multi-step mutagenesis. The mutant produced an appreciable yield of glucoamylase in the culture medium. The enzyme was purified and was shown to be homogeneous. It hydrolysed successive glucose residues from the non-reducing end of starch molecules. The purified enzyme had an optimum pH of 5·0 and was stable over the pH range 3·0–7·0. It was highly active over a broad temperature range, 30–75°C, with optimal activity at 60°C. The molecular weight was 70 000 as determined by Sephadex G-200 filtration. The enzyme showed a decrease in K _m values with increasing chain length of the substrate molecule.     

Isolation and growth physiology of novel thermoactinomycetes

A total of 34 thermophilic isolates identified as members of the genus Thermoactinomyces by a range of chemotaxonomic, microscopic and determinativebiochemical tests, were isolated from two acid soils. Growth studies in shake flask and fermenteridentified the isolates to be moderately acidophilic with growth occurring between pH4·5 and 6·0 with optima at pH 5·0. The isolates differed considerablyfrom known Thermoactinomyces cultures in their pH profile, colony morphology andin several biochemical tests.Extracellular enzyme activities are identified and partiallycharacterized in termsof their thermostability, pH and temperature profiles from crude supernatantfluid samples. Optimal protease, amylase and pullulanase activity was observed at pH5·0–5·5 and 75–80 °C with each showing T ₍₅₀₎ values of 10, 30 and 30 min, respectively. A highly thermotolerant extracellularesterase was also identified which retained 50% activity after 8 h at 90°C . This is the firstreport of an acidophilic member of the genus Thermoactinomyces.     

Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase-producing alkaliphilic Bacillus sp. AR-009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. WN11

A thermostable amylase-producing Bacillus sp. WN11 was isolated from Wondo Genet hot spring in Ethiopia. The enzyme had a temperature optimum of 75–80 °C. Over 80% of its peak activity was in the pH range of 5–8, with an optimum at 5·5. Thermal stability of the enzyme at 105 °C was higher with the addition of starch. The stabilizing effect of starch was concentration-dependent, showing better stability with increasing concentration of starch. At liquefying temperature (105 °C), addition of Ca²⁺ did not result in further improvement of the stabilizing effect of starch. This indicates that in the presence of starch, WN11 amylase does not require Ca²⁺ as a stabilizer at liquefying temperatures as high as 105 °C.     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

CMC-liquefying enzyme, a low molecular mass initial cellulose-decomposing cellulase responsible for fragmentation from Streptomyces sp. LX

Streptomyces sp. LX, newly isolated from soil, was shown to secrete a carboxylmethylcellulose (CMC)-liquefying enzyme that cleaves the CMC chains, releasing negligible reducing terminals. The new enzyme, named component C₂, was purified to homogeneity by dialysation. It has a molecular mass of 9·8 kDa. The pH optimum of the enzyme activity is 6·4 and its temperature optimum is 50°C. It retains full activity at pH 4–6·4 upon incubation at 50°C for 30 min. The enzyme has significant fragmentation activity on filter paper despite the absence of weight loss, release of reducing sugars and depolymerization during incubation with filter paper. The one-electron oxidative reaction is shown not to participate in the fragmentation of filter paper by enzyme C₂.     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。     

Daily ration and prey size of juvenile piscivore Japanese Spanish mackerel

Daily ration of juvenile Japanese Spanish mackerel Scomberomorus niphonius (32·1–33·1 mm standard length, L _S) was estimated at three temperatures (18·6, 19·5 and 21·2° C) in the laboratory. Gastric evacuation rate ranged between 0·398 (18·6° C) and 0·431 (21·2° C). Diel change in instantaneous consumption rate indicated that juvenile Japanese Spanish mackerel are daylight feeders. The estimated values of the daily ration ranged between 66·1%(18·6° C) and 82·6%(21·2° C) of body mass. These per cent values of daily ration were converted to daily consumption in mg (28 mg at 18·6° C to 34 mg at 21·2° C) using the mean dry body mass of juvenile Japanese Spanish mackerel of 30 mm (42·1 mg). Stomach content analysis of wild specimens collected in the Seto Inland Sea, south‐western Japan, revealed that the majority of wild Japanese Spanish mackerel larvae and juveniles ingested fish larvae with a body size >50% of the predator L _S. Based on the predator‐prey size relationship, the daily consumption of a Japanese Spanish mackerel juvenile of 30 mm was equivalent to 5·1 (18·6° C) to 6·4 (21·2° C) Japanese anchovy Engraulis japonicus larvae which was the dominant prey organism in stomachs of the wild Japanese Spanish mackerel.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.     

Study of pectin esterase and changes in pectin methylation during normal and abnormal peach ripening

Peach fruit ( Prunus persica cv. Hermosa) were allowed to ripen immediately after harvest or after 30 days of 0°C storage. The fruits lost 75–80% of their firmness after 5 days at 20°C. During ripening after harvest there was a loss of both uronic acid and methyl groups from the cell wall. Cell wall labelling with JIM 7, a monoclonal antibody which recognized pectins with a high degree of methylation, was lower in ripe fruits than in freshly harvested fruits. However, ripe fruit cell walls did not cross-react with JIM 5, which recognizes pectins with low methylation. During storage, de-methylation occurred and in fruit ripened after storage there was little further change in pectin methylation or pectin content in the cell walls. The labelling of stored or stored plus ripened cell walls with JIM 7 was similar, but the cell walls of fruit ripened after storage showed some low cross-reactivity with JIM 5. The in vitro activity and mRNA abundance of pectin esterase (EC 3.1.1.11) was not correlated with the amount of de-esterification as measured chemically or by immuno-labelling in the cell walls. Eighty percent of the fruits which ripened after storage developed a woolly texture. It is suggested that woolliness is due to de-esterification of pectins, not accompanied by depolymerization, which leads to the formation of a gel-like structure in the cell wall.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

The effect of temperature and fish size on growth and feed efficiency ratio of juvenile spotted wolffish Anarhichas minor

The growth properties of juvenile spotted wolffish Anarhichas minor reared at 4, 6, 8 and 12° C, and a group reared under 'temperature steps', (T‐step) i.e . with temperature reduced successively from 12 to 9 and 6° C were investigated. Growth rate and feed efficiency ration was significantly influenced by temperature and fish size. Overall growth rate was highest at 6° C (0·68% day⁻¹) and lowest at 12° C (0·48% day⁻¹), while the 4 and 8° C, and the T‐step groups had similar overall growth rates, i.e . 0·59, 0·62 and 0·51% day⁻¹ respectively. Optimal temperature for growth ( T _{opt G} ) and feed efficiency ratio (T_{opt FCE}) decreased as fish size increased, indicating an ontogenetic reduction in T _{opt G} and T _{opt FCE}. The results suggest a T _{opt G} of juvenile spotted wolffish in the size range 135–380 g, dropping from 7·9° C for 130–135 g to 6·6° C for 360–380 g juveniles. The T _{opt FCE} dropped from 7·4° C for 120–150 g to 6·5° C for 300–380 g juveniles. A wider parabolic regression curve between growth, feed efficiency ratio and temperature as fish size increased, may indicate increased temperature tolerance with size. Individual growth rates varied greatly at all time periods within the experimental temperatures, but at the same time significant size rank correlations were maintained and this may indicate stable size hierarchies in juvenile spotted wolffish.     

The filamentous fungus Aspergillus nidulans produces an α-L-rhamnosidase of potential oenological interest

The production of α- l -rhamnosidase by Aspergillus nidulans has been investigated. In the presence of rhamnose as sole carbon source, this fungus produces an α- l -rhamnosidase of molecular weight 90 kDa. Production of this enzyme is under carbon catabolite repression, apparently by a CreA-independent system. At acidic ambient pH there is an increase in the synthesis of the enzyme which is not related to PacC. Using ρ-nitrophenyl-α- l -rhamnopyranoside as substrate, the enzyme activity in culture filtrates shows pH and temperature optima of 4·5–8 and 40–50 °C, respectively. At the concentrations found in must or wine, enzyme activity was only slightly affected by glucose and SO₂ and partly inhibited by ethanol, indicating a potential for use in wine aroma release.     

Phytase activity of Selenomonas ruminantium: a preliminary characterization

Obligately anaerobic ruminal bacteria have been found to possess phytase activity, in particular, Selenomonas ruminantium . The phytase activity of S. ruminantium JY35 was produced late in growth and required neither phytate for induction nor phosphate limitation for derepression. The activity was completely cell-associated with a significant fraction extractable by a magnesium chloride solution. Zymogram analysis suggested that the activity was the result of a single gene product of a monomeric nature and approximately 46 kDa in size. The phytase had a temperature optimum of 50–55 °C, but activity dropped off sharply at 60 °C. Phytase activity was optimal over the pH range of 4·0–5·5, and dependent on the nature of the buffer used. Activity was inhibited by citric acid buffer and by the addition of 5 mmol l⁻¹ Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Hg²⁺. The addition of 5 mmol l^–1 Pb²⁺ to the enzyme assay appeared to enhance activity of the enzyme.