Isolation of a bioemulsifier from Candida lipolytica

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Purification and Characterization of Liposan, a Bioemulsifier from Candida lipolytica

The inducible water-soluble bioemulsifier liposan (M. C. Cirigliano and G. M. Carman, Appl. Environ. Microbiol. 48:747-750, 1984) was purified from the yeast Candida lipolytica. The purification procedure included repeated solvent extractions of a concentrated culture filtrate and Affi-Gel concanavalin A affinity chromatography. The procedure yielded a preparation containing a major band (M_r = 27,600) which stained for protein and carbohydrate upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Liposan is composed of approximately 83% carbohydrate and 17% protein. Acid and enzymatic digestions of the emulsifier revealed that the carbohydrate portion is a heteropolysaccharide consisting of glucose, galactose, galactosamine, and galacturonic acid. Liposan effected and stabilized oil-in-water emulsions with a variety of commercial vegetable oils. Emulsification and stabilization properties of liposan were compared to those of a number of commercial emulsifiers and stabilizers.     

Factors affecting the consumption of 2,3-butanedione by Saccharomyces cerevisiae

Production and degradation of diacetyl by a commercial Saccharomyces cerevisiae strain was studied. This yeast did not produce diacetyl but could consume it. Diacetyl degradation activity was biological and was present even when the yeast was grown in the absence of diacetyl. Maximum specific activity was obtained when the yeast was grown in 280 μmol of diacetyl, 1 vvm of aeration and 37°C.     

Enzymes of Candida albicans cell-wall lytic system produced by Streptomyces thermodiastaticus

The production of the enzymes of Candida albicans cell-wall lytic system by S. thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest lytic activity was obtained after 18 h of incubation at pH 5.5 and an incubation temperature of 50 degrees C. The carbon source influenced the production of the enzymes of the yeast cell wall lytic system. Maximum lytic activity was obtained when Candida albicans cell-wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml level was the best nitrogen source for the biosynthesis of the enzymes of the yeast lytic system. From all phosphor sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/l) and Tween 80 (0.1%), respectively were found to favour highest enzymes production of the lytic system. The Candida albicans cell-wall lytic system produced by S. thermodiastaticus mainly contained chitinolytic and proteolytic activities.     

Bioemulsifier production by Bacillus stearothermophilus VR-8 isolate

Bacillus stearothermophilus produced an extracellular bioemulsifier during growth in a medium containing 4% crude oil. Over the temperature range of 45° to 70°C, maximum recovery (0·6 g 1^-1) occurred at 50°C. The emulsifier had its greatest activity on benzene, among the hydrocarbons tested. Acetone precipitated, dialysed emulsifier contained 46% protein, 16% carbohydrate and 10% lipid. The emulsification activity was stable over a broad range of temperature (50–80°C), pH (2–8) and salt concentration (5% NaCl, 5% CaCl₂ and 1% MgCl₂). Thus, this emulsifier was found to be better than liposan (showing emulsifying activity between pH 2–5 and stable up to 70°C) in terms of pH and temperature stability. Additionally, it was also salt tolerant, suggesting its potential use in crude oil tank clean-up and enhanced oil recovery.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Production of Lactase by Candida pseudotropicalis Grown in Whey

Lactase (beta-d-galactosidase) was produced by Candida pseudotropicalis grown in deproteinized whey. Maximum enzyme production in 2% whey was obtained by supplementation with 0.15% yeast extract, 0.1% (NH(4))(2)SO(4), and 0.05% KH(2)PO(4) (wt/vol). Highest enzyme values (4.35 U/mg of cells and 68 U/ml) were obtained with 10 to 12% whey, while enzyme yield was maximal in 2% whey (0.87 U/mg of whey). Optimal initial pH for cultivation was 3.5. The best conditions for extraction included 2% (wt/vol) chloroform, 10 h of treatment, pH 6.6 and higher, and 30 to 37 degrees C. Optimum pH and temperature for enzyme activity were 6.2 and 47 degrees C. The enzyme had a K(m) for O-nitrophenyl-beta-d-galactopyranoside of 3.06 x 10 M and the initial V(max) was estimated as 6.63 x 10 M per min. It hydrolized 50 and 100% of the lactose in whey and milk within 4 and 5 h, respectively, at 37 degrees C. The lyophilized enzyme retained 95% of activity for 3 months when stored at -20 degrees C.     

Studies on cellulase production by a Bacillus subtilis

Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 °C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 °C, and the enzyme was highly stable up to 60 °C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.     

枯草芽孢杆菌中性β—甘露聚糖酶的产生及性质

由土壤中分离出一株产中性β甘露聚糖酶的枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）,编号ＢＭ９６０２。该菌在液体培养条件下,产生中性β甘露聚糖酶。多糖能作为碳源,而单糖不能作为碳源;有机氮源优于无机氮源。产酶最适培养基组成：魔芋粉４％,牛肉蛋白胨和酵母膏各１％。产酶最适培养条件：培养基起始ｐＨ８５,３５℃,振荡培养３６ｈ。以槐豆胶为底物,培养滤液中性β甘露聚糖酶活力为９６ＩＵ／ｍＬ。酶在ｐＨ５０～１００和５０℃下稳定;作用最适条件为ｐＨ６０和５０℃;水解魔芋粉和槐豆胶均产生寡聚糖。     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Optimization of extracellular thermotolerant alkaline protease produced by marine <Emphasis Type="Italic">Roseobacter</Emphasis> sp. (MMD040)

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.     

A highly thermostable and alkaline amylase from a Bacillus sp. PN5

A highly thermostable alkaline amylase producing Bacillus sp. PN5 was isolated from soil, which yielded 65.23 U mL(-1) of amylase in medium containing (%) 0.6 starch, 0.5 peptone and 0.3 yeast extract at 60 degrees C, pH 7.0 after 60 h of incubation. Maximum amylase activity was at pH 10.0 and 90 degrees C. The enzyme retained 80% activity after 1 h at pH 10.0. It exhibited 65% activity at 105 degrees C and had 100% stability in the temperature range between 80 and 100 degrees C for 1 h. In addition, there was 86.36% stability after 1-h incubation with sodium dodecylsulphate. These properties indicated possible use of this amylase in starch saccharification and detergent formulation.     

L-Asparaginase Synthesis by Erwinia aroideae

Maximum L-asparaginase activity was obtained when 1.0% lactose and 1.5% yeast extract were supplied as carbon and nitrogen sources, respectively. Glucose inhibited the enzyme formation. The diauxie phenomenon was observed with Erwinia aroideae NRRL B-138 grown in a medium containing glucose and lactose.     

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.     

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.     

Purification and characterization of maltose phosphorylase from Bacillus sp. RK-1

Bacillus sp. RK-1 was isolated as a bacterium that produced maltose phosphorylase (MPase) in the culture supernatant. Screening was done from among about 400 isolates that could grow at 55 degrees C in a medium containing maltose as the sole carbon source. The enzyme was purified to an electrophoretically homogeneous state and some properties were investigated. The Mr of the enzyme was estimated to be 170 kDa by gel filtration and 88.5 kDa by SDS-PAGE, suggesting that it consisted of two identical subunits. The enzyme showed optimum activity around pH 6.0-7.0 and the optimum temperature was about 65 degrees C. The enzyme was stable in the range of pH 5.5-8.0 after keeping it at 4 degrees C for 24 h and retained the activity up to about 55 degrees C after keeping it for 15 min. This is the first report about an MPase that could be produced in the culture supernatant. Furthermore, these investigations showed that this MPase is one of the most thermostable ones reported so far.     

Mycelium-bound carboxylesterase from Aspergillus oryzae: an efficient catalyst for acetylation in organic solvent

Dry mycelium of a strain of Aspergillus oryzae efficiently catalyzed the esterification between free acetic acid and primary alcohols (geraniol and ethanol) in organic solvent. The growth conditions to obtain high activity of mycelium-bound enzymes were firstly evaluated. A medium containing Tween 80 as carbon source furnished mycelium with the highest activity in the hydrolysis of alpha-naphthyl esters (alpha-N-acetate, butyrate, caprylate). Dry mycelium was employed to select suited conditions for an efficient acetylation of ethanol and geraniol in heptane. Maximum productions were obtained using 30 g l(-)(1) of lyophilized cells: 12.4 g l(-)(1) of geranyl acetate were produced at 80 degrees C starting from 75 mM geraniol and acetic acid (84% molar conversion) and 4.1 g l(-)(1) of ethyl acetate at 50 degrees C from 50 mM ethanol and acetic acid (94% molar conversion) after 24 h. The stability of the mycelium-bound carboxylesterases are notable since only 10-30% loss of activity was observed after 14 days at temperatures between 30 and 50 degrees C.     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035.

Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.