Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5–2.3 h (22°C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72–96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28°C, but low levels of amylase were observed in TSBWD cultures grown at 19–23°C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for -amylase. Activity was maximum on soluble starch at a temperature between 40°C and 50°C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

Properties of an extracellular amylase purified from a Bacillus species

A strain of Bacillus produced an amylase with properties characteristically different from known bacterial amylases. The purified 80 kDa protein of pI 5.1 dextrinized starch, glycogen and pullulan. The temperature and pH optima of the enzyme were 60 °C and 6.6 respectively. In the presence of 0.05 M CaCl₂, the enzyme retained stability for 15 min at 80 °C. Antibodies raised to the amylase protein showed no reaction with -amylases of Bacillus sp. and B. licheniformis. In culture, proteolytic degradation of the enzyme was observed.     

Short communication: Thermostability of α-amylase produced by Bacillus sp. E2—a thermophilic mutant

An -amylase from a hyper-producing strain of Bacillus (sp. E2) was stable at 70°C for 30 min but was quickly inactivated at higher temperatures. In the presence of 10mm Ca²⁺ and starch (20% w/v), however, the enzyme was stable at 90°C for 10 min and after 30 min at 100°C still retained 26% of its initial activity.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)--glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and -glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Production kinetics and stability properties of ϖ(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase is a key enzyme that channels primary metabolites to a tripeptide common to cephalosporin and cephamycin biosynthesis inStreptomyces clavuligerus. Time-course studies indicated that theS. clavuligerus ACV-synthetase was stable during the cephamycin C fermentation: the enzyme was produced early in the growth phase and its activity remained high up to 96 h of growth. The detection of crude ACV-synthetase activity in older cultures was best achieved with an assay medium supplemented with 5 mM phosphoenolpyruvate, at lower ATP concentrations. During storage at 4°C, a progressive decrease in the stability of crude ACV-synthetase was observed with increasing culture age. Although a proteinolytic activity with a pH optimum at 8.2 was detected in crude cell-free extracts, no significant variation was observed in its activity with increasing culture age to account for the instability of ACV-synthetase in vitro. Addition of proteinase inhibitors did not improve the stability of the enzyme. However, a stabilization cocktail containing dithiothreitol. MgCl₂, the three substrate amino acids, and glycerol increased the stability of the enzyme isolated from cultures grown for 30–40 h, which was shortly after the appearance of antibiotics in the culture fluid. This stabilized enzyme retained half of its initial activity after 6 days at 4°C.     

Spray drying of the dehalogenating bacterium Rhodococcus sp.

The bacteria Rhodococcus sp. and Xanthobacter autotrophicus have the ability to dehalogenate a broad range of halogenated hydrocarbons. The applicability of spray drying to the preservation of the microorganisms and the intracellular enzyme halidohydrolase (E.C.3.8.1.1) was examined. K₂SO₄, MgSO₄, glutamate and sucrose were added as stabilizers and carriers. Spray drying was carried out at inlet air temperatures of 100–120 °C and outlet air temperatures of 65–72 °C. Best results were obtained by the addition of 5% K₂SO₄ and at 107 °C air inlet temperature. Dried preparations of Rhodococcus sp. exhibited a crystalline consistency and a 95% recovery of cellular activity. After storage at 4 °C for six months the enzyme preparation showed no loss in activity. Spray dried preparations of Xanthobacter autotrophicus showed only a 4% recovery of cellular activity.List of Symbols MSG Monosodiumglutamate - RC % Recovery of stabilizer and biomass - RCA % Recovery of cellular activity ([U/g biomass after the spraydrying]/[U/g biomass of untreated cells]) 100 - RCB % Recovery of biomass - SR % Survival rate - T ₁ °C Inlet air temperature - T ₂ °C Exit air temperature - W % Water content - Y.Akt % Yield in enzyme activity This work was supported by the Jubiläumsfond der Oesterreichischen Nationalbank, Projekt No. 4499.     

Production of xylanase by Emericella nidulans NK-62 on low-value lignocellulosic substrates

Thermotolerant Emericella nidulans NK-62 was isolated from bird nesting material and was tested for its ability to produce xylanase. The fungus when grown on a medium containing wheat bran (2% w/v) supplemented with Czapek's mineral salt solution at 45 °C for 7 days produced 362 IU/ml of xylanase (EC 3.2.1.8). The specific activity of E. nidulans NK-62 xylanase was found to be 275 IU/mg of total protein. The enzyme was found to be active over a broad temperature and pH range with 60 °C as optimum temperature for enzyme activity. The enzyme was stable at 50 °C and its half-life at 55 °C was 45 min. -xylosidase (EC 3.2.1.37) and carboxymethylcellulase (EC 3.2.1.4) activities, 0.018 and 0.21 IU/ml respectively, were also noticed. The fungus was screened for its ability to produce xylanase on four different lignocellulosic substrates. It produced 318.9 IU/ml of cellulase-free xylanase on corn cobs. The fungus could also utilize lentil bran (seed husk of Lens esculentus) and meal of groundnut shells to produce 84.8 and 17.3 IU/ml xylanase respectively.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Amylolytic activity of Thermomonospora fusca

Amylases which produce maltotriose as the major end-product from starch are relatively rare. The thermophilic actinomycete, Thermomonospora fusca, produced an extracellular -amylase which generated maltotriose as 61% of the identified products. The addition of maltotriose to a glucose-adapted exponential phase culture at 55°C in mineral salts medium caused rapid induction of amylase biosynthesis. Addition of glucose to cells growing on starch did not repress amylase biosynthesis because the actinomycete had a marked preference for maltotriose over glucose. The pH and temperature optima for the amylase activity of concentrated, washed extracellular protein were 6.0 and 65°C, respectively, with an energy of activation of 59kJ/mol. The thermostability of the concentrated, washed amylase was increased by the presence of its starch reaction products, but not by added Ca2+.     

A procedure for preservation of the mosquito pathogen Culicinomyces clavisporus

Summary An effective storage procedure has been demonstrated for the mosquito pathogen Culicinomyces clavisporus. A mycelial preparation was harvested by filtration, sprayed with a sucrose solution and air dried at 20°C in a laminar flow cabinet until the mycelial mat became crisp. This material was then ground in a hammer mill and particles of less than 355 m were sieved out. Viability of the particles was assessed by studying mycelial growth and conidial formation when particles were added to water agar plates or to water. The particles retained 100% viability after 9 weeks storage at-20°C or 6 days at 4°C. Preparations lost activity rapidly if stored at 20°C. Conidia produced by this method were pathogenic to mosquito larvae.     

Fusion of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> leucine aminopeptidase II with the raw-starch-binding domain of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 -amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70°C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k _cat value.