Keratinase Production by Newly Isolated Antarctic Actinomycete Strains

Summary The ability of actinomycete strains newly isolated from Antarctic soils to produce keratinolytic enzymes during growth on sheep wool waste was investigated. The strains which displayed highest keratinase activity and identified as Streptomyces flavis 2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic) were selected for a more detailed analysis. The addition of starch to the growth medium affected keratinase secretion by both strains. After 5 days of cultivation, a 6-fold increase in keratinase activity of strain 11A was observed in the presence of 11 g starch/l and a 9-fold increase in keratinase activity of the strain 2BG in the presence of 5 g starch/l. The results obtained showed that both newly isolated strains are very promising for effective processing of native keratinous wastes. To our knowledge, this is the first report of Antarctic actinomycete strains that were able to grow on keratin-containing wastes by producing keratinolytic enzymes.     

津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定

目的:从津巴布韦烟叶中分离产蛋白酶菌和产淀粉酶能力最高的菌株,并对其进行鉴定。方法:采用淀粉富集培养基和酪蛋白富集培养基分别分离津巴布韦烟叶中的产淀粉酶和产蛋白酶菌株,通过生理生化实验和16SrRNA序列分析鉴定分离的菌株。结果:产蛋白酶菌株菌体不透明、表面有褶皱,蛋白酶酶活为52.10±0.13 U/mL;产淀粉酶菌株菌体表面呈黏状,淀粉酶酶活为3.69±0.07 U/mL;产蛋白酶与产淀粉酶的2株菌均与枯草芽孢杆菌的16S rRNA序列有100%的相似性,结合生理生化指标初步鉴定为枯草芽孢杆菌。结论:获得的2株菌在降解烟叶的蛋白质和淀粉过程中可能起重要作用。     

Screening for and identification of starch-, amylopectin-, and pullulan-degrading activities in bifidobacterial strains

Forty-two bifidobacterial strains were screened for alpha-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.     

Activity and cellular localization of amylases of rabbit cecal bacteria

Five 11-week-old rabbits, fed a commercial granulated feed, were slaughtered and cecal starch-degrading bacteria enumerated; total concentration of cultivable bacteria utilizing starch averaged 5.5 x 10(10) CFU/g. The activity and cellular localization of amylases was determined in 9 bacteria identified as Actinomyces israeli (strains AA2 and AD4), Bacteroides spp. (strain AA3), Dichelobacter nodosus (strain AA4), Mitsuokella multiacidus (strain AA6), Eubacterium spp. (strains AA7 and AB2), Clostridium spp. (strains AD1 and AA5). Four strains (AA3, AA4, AA5, AD4) produced extracellular amylases with an activity of 26-35 micromol of reducing sugars per h per mg of protein; in five strains (AA2, AA6, AA7, AB2, AD1) amylases were membrane-bound with an activity of 14-18 micromol of reducing sugars per h per mg of protein. All strains exhibited a low intracellular amylolytic activity. The pH optimum of amylases was 6.8-7.0. In strains producing extracellular amylases a substantial loss of viscosity was observed during incubations of cultivation supernatant with starch, similar to viscosity reduction in starch solutions treated with alpha-amylase; this indicates an endo-type (random cleavage) of extracellular amylase reaction in the bacteria under study. No strain possessed glucoamylase activity.     

The protective effects of high amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in the mouse intestinal tract

The possibility of using high amylose maize starch granules as a delivery system for probiotic bacteria has been investigated using Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B which were isolated from a healthy human. The Bifidobacterium cells were able to adhere to the amylomaize starch granules and were also able to hydrolyse the starch during growth. Initially, in vitro studies were carried out by studying the survival of strains Bifidobacterium LaftiTM 8B and LaftiTM 13B when exposed to pH 2.3, 3.5 and 6.5 as well as 0.03 and 0.05% w/v bile acids. Both strains were grown either in the absence or presence of high amylose maize starch granules, then mixed with the high amylose maize starch granules and exposed to acidic buffers or bile acid solutions. It was shown that growth in and the presence of high amylose maize starch granules led to enhanced survival of strains LaftiTM 8B and LaftiTM 13B. Subsequently, survival in vivo was monitored by measuring the faecal level of Bifidobacterium LaftiTM 8B after oral administration of the strain to mice. A sixfold better recovery of strain LaftiTM 8B from mice faeces after oral dosage was noted for cells grown in amylose-containing medium compared with controls. It was concluded that high amylose maize starch granules contributed to enhanced survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B.     

Modified α-amylase activity among insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, α-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (≥70-fold purification, ≤19% yield). Single α-amylase bands with the same molecular mass (53.7 kDa) were revealed for each insect strain. Higher activity was obtained at 35-40 °C and at pH 5.0-7.0 for all of the strains. The α-amylase from the resistant no-cost strain exhibited higher activity towards starch and lower inhibition by acarbose and wheat amylase inhibitors. Opposite results were observed for the α-amylase from the resistant cost strain. Although the α-amylase from the resistant cost strain exhibited higher affinity to starch (i.e., lower K_m), its V_max-value was the lowest among the strains, particularly the resistant no-cost strain. Such results provide support for the hypothesis that enhanced α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance.     

Genetically determined electrophoretic variants of the major urinary protein (Mup) complex in mouse urine

The major urinary protein (Mup-complex) excreted in mouse urine, has been studied electrophoretically both on starch gel and on cellogel. On stargel six anodally migrating protein bands were observed. These bands are designated component 3, 2', 1, and 4 (i.e. two bands) in the order of decreasing mobility toward the anode. The slower protein band of component 4 on starch gel was not observed on cellogel. By testing mouse inbred strains, we were able to dinstinguish five male and four female Mup phenotypes. Test crosses suggested a four-allelic (a, b, c, d,) variation with regard to components 2', 2 and 1: 'group A' strains showed component 1, 'group B' strains components 1 and 2, 'group C' and 'group F' strains none, and 'group D' strains showed components 1 and 2'. Component 3 may be encoded by another Mup locus, although no crossing-over has been observed: presence (A, B, D, and F strains), absence (C strains). Insufficiently reproducible demonstration of the variation with regard to component 4, forced us to exclude this component for strain distinction. The Mup phenotypes described, can be useful for the detection of certain strain contaminations, especially if F1 hybrid Mup phenotypes are distinguishable.     

Efficient production of ethanol from raw starch by a mated diploid Saccharomyces cerevisiae with integrated α-amylase and glucoamylase genes

The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae, which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3′-half of the α-agglutinin gene as the anchor protein, and the α-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67, 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process.     

Screening for and Identification of Starch-, Amylopectin-, and Pullulan-Degrading Activities in Bifidobacterial Strains

Forty-two bifidobacterial strains were screened for α-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.     

Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer,using a kar1 mutant as vector

Summary A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an autotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; ( ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and the, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.     

八株芽孢杆菌菌株的分类及固氮活性的测定

从8个省市的不同土壤分离了200个菌株,筛选出8株（JR₁,JR₂,JR₅,ZZ₃,ZZ₁₂,ZZ₁₆,M₁,K₄）能在无氮培养基上生长良好的典型的芽抱杆菌,其中6株（JR₁,JR₂,JR₄和ZZ₃,ZZ₁₂,ZZ     

Genetically determined electrophoretic variants of the major urinary protein (Mup) complex in mouse urine

The major urinary protein (Mup-complex) excreted in mouse urine, has been studied electrophoretically both on starch gel and on cellogel.
On stargel six anodally migrating protein bands were observed. These bands are designated component 3, 2', 2, 1, and 4 (i.e. two bands) in the order of decreasing mobility toward the anode. The slower protein band of component 4 on starch gel was not observed on cellogel.
By testing mouse inbred strains, we were able to dinstinguish five male and four female Mup phenotypes. Test crosses suggested a four-allelic ( a, b, c, d, ) variation with regard to components 2', 2 and 1: 'group A' strains showed component 1, 'group B' strains components 1 and 2, 'group C' and 'group F strains none, and 'group D' strains showed components 1 and 2'. Component 3 may be encoded by another Mup locus, although no crossing-over has been observed: presence (A, B, D, and F strains), absence (C strains). Insufficiently reproducible demonstration of the variation with regard to component 4, forced us to exclude this component for strain distinction.
The Mup phenotypes described, can be useful for the detection of certain strain contaminations, especially if F₁ hybrid Mup phenotypes are distinguishable     

Improved Method for Detection of Starch Hydrolysis

A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and α-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of α-amylase activities in culture filtrates or column effluents.     

Direct conversion of starch to L(+) lactic acid by amylase producing Lactobacillus amylophilus GV6

Lactobacillus amylophilus strain GV6, isolated from corn starch processing industrial wastes, was amylolytic and produced 0.96?g L(+) lactic acid per gram of soluble starch. The optimum temperature and pH for growth and L(+) lactic acid production were 37?°C and 6.5, respectively. At low substrate concentrations, the lactic acid production on corn starch was almost similar to soluble starch. The strain is fermenting various naturally available starches directly to lactic acid. The total amylase activity of the strain is 0.59?U/ml/min. The strain produced 49 and 76.2?g/l L(+) lactic acid from 60?g/l corn starch and 90?g/l soluble starch, respectively. This is the highest L(+) lactic acid among the wild strains of L. amylophilus reported so far.     

Influence of the carbon source on the growth and lignocellulolytic enzyme production by Morchella esculenta strains

Growth and lignocellulolytic enzymes production by two Morchella esculenta strains (BAFC 1728 and BEL 124) growing in solid state fermentation using different lignocellulosic materials along 58 days was characterized. Both strains were able to grow on the three substrates: wheat bran, wheat bran plus corn starch, and rolled oat. The growth was characterized by measuring chitin content, reducing sugars, pH, dry weight loss, and extractable proteins, such parameters varied substantially with substrate and strain used. The maximum rate of growth in both strains was observed between 5 and 28 days. Regarding enzyme production, as a general trend strain BAFC 1728 produced the highest titres. The most evident difference was observed in laccase production by this strain on wheat bran, which exceeded that observed in strain BEL 124 by tenfold (7.45 U g⁻¹).     

Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining δ-integration and polyploidization through cell fusion. Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase/α-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The α-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.     

Protein enrichment of potato processing waste through yeast fermentation

Potato starch obtained from waste waters of chips manufacturing was used as a fermentation substrate for yeast protein enrichment. Among 18 yeast strains, 6 strains were screened according to their biomass yield and protein content after fermentation for 16 h at 30 degrees C in an aerated glucose-based liquid media (4.5 Ls). Using concentrated media (25% solids) made from potato starch pre-hydrolyzed with malt flour and batch-fermented for 20 h at 26 degrees C under aerobic conditions, Candida utilis ATCC 9256 was the most efficient protein-forming strain. Scaled-up at the 100 Ls level, the aerobic batch process was improved under fed-batch conditions with molasses supplementation. After drying, fermented starch contained 11-12% protein, including 7-8% yeast protein.     

Study of the carbohydrate content in the mycelia of an active strain producing oxytetracycline and in an inactive mutant]

The content of carbohydrates in the mycelium of the active strain and inactive mutant of the oxytetracycline-producing organism under conditions favourable (starch medium) and unfavourable (glucose medium) for the antibiotic biosynthesis was studied. The mycelium of both organisms was fractionated and carbohydrate distribution according to the mycelium fractions and carbohydrate content in every fraction were investigated. No significant differences were observed between the active strain and inactive mutant with respect to the characteristics studied. The carbon source in the medium had the dominating effect on the chemical composition of the mycelium. The mycelium of both strains grown on the starch medium contained much more carbohydrates than that grown on the glucose medium. The carbohydrates of the mycelium grown on the starch medium were mainly found in fraction III and must be represented by polysaccharides.     

Nutritional requirements and simplified cultivation medium to study growth and energetics of a sourdough lactic acid bacterium Lactobacillus fermentum Ogi E1 during heterolactic fermentation of starch

AIMS: Nutritional requirements of Lactobacillus fermentum Ogi E1 were studied in order to define a simplified fermentation medium. METHODS AND RESULTS: When grown with MRS-medium in 2l bioreactors, a biphasic pattern of growth and metabolite production was observed. Study of nutritional requirements resulted in a simplified medium (SYAM) that allowed, under anaerobiosis, similar results to be obtained as in MRS medium, but without biphasic fermentation kinetics. The best substrates for both growth and amylase production were starch and maltose. Although melibiose, raffinose, fructose, sucrose and glucose also supported growth, lower amylase activity was observed. CONCLUSION: The physiology of the strain can be investigated with SYAM medium, using either starch or maltose as substrate. The strain also presented potential for alpha-galactoside fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus fermentum was one of the dominant bacteria of African maize dough fermentations. Amylolytic strains with activity against other compounds (i.e. raffinose) suggested a potential to be used as starter for cereal fermentation.     

Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148.

An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.