Study of thermostable chitinases from Oerskovia xanthineolytica NCIM 2839

The mesophilic organism, Oerskovia xanthineolytica NCIM 2839, was adapted to grow at moderate thermophilic temperatures. At these elevated temperatures, it was found to produce two thermostable chitinases—C1 and C2. These were purified by ion exchange chromatography using DEAE cellulose. The chitinases C1 and C2 were found to be stable in a pH range from 3.0 to 9.0 with 7.5 and 8.0 being the optimum pH, respectively. The optimum temperatures of the activities of C1 and C2 were 50 and 55°C, respectively. These were activated by Mn⁺⁺ and Cu⁺⁺and inactivated by Hg⁺⁺. This is first report of an extracellular thermostable chitinase being produced by O. xanthineolytica NCIM 2839.     

Efficient conversion from polysialogangliosides to monosialotetrahexosylganglioside using Oerskovia xanthineolytica YZ-2

A new sialidase-producing strain isolated from soil was identified as Oerskovia xanthineolytica YZ-2. Sialidase was produced when Oerskovia xanthineolytica YZ-2 was exposed to polysialogangliosides. The sialidase of Oerskovia xanthineolytica YZ-2 hydrolyzed sialic acid linkages in polysialogangliosides, and released monosialotetrahexosylganglioside (GM1). The sialidase had the capability of product specificity because it did not attack the sialic acid linkage in GM1. Therefore, Oerskovia xanthineolytica YZ-2 was used for GM1 production from polysialogangliosides. In flasks cultivation phase, it was proved that Oerskovia xanthineolytica YZ-2 could convert polysialogangliosides to GM1 efficiently. Scaling-up the bioprocess with 8% crude ganglioside, polysialogangliosides was converted to GM1 by Oerskovia xanthineolytica YZ-2 in 30 L bioreactor after 18 h. The relative content of GM1 increased from 16.3% in crude ganglioside to 83.7% after Oerskovia xanthineolytica YZ-2 conversion. Therefore, a simple, large-scale conversion process for GM1 production from polysialogangliosides was achieved using Oerskovia xanthineolytica YZ-2 as a biocatalyst.     

Cellular fatty acid composition of Oerskovia species,CDC Coryneform groups A-3, A-4, A-5, Corynebacterium aquaticum,Listeria denitrificans and Brevibacterium acetylicum

Twenty four strains representing eight species of gram positive yellow-pigmented rods (Oerskovia turbata, Oerskovia xanthineolytica, CDC Coryneform groups A-3, A-4, A-5, Listeria denitrificans, Corynebacterium aquaticum and Brevibacterium acetylicum) were divided into two major groups based on the relative amounts of 12 methyltetradecanoate (15:0^a) obtained by capillary gas liquid chromatography. O. turbata, O. xanthineolytica, CDC groups A-3 and A-4, L. denitrificans and C. aquaticum were placed in the first group due to the presence of a higher percentage (29–47%) of 15:0^a, than CDC group A-5 and B. acetylicum. The latter contained 2–6% of this fatty acid, and were placed in the second group.All species in the two groups except C. aquaticum and CDC group A-4, were further separated from each other based on the qualitative and quantitative differences in their fatty acid compositions. In addition, the eight strains of CDC group A-5 revealed four different patterns and were further divided into four subgroups. This study supports the importance of the composition of cellular fatty acids in differentiating some closely related organisms.     

High level phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 in solid state culture: response surface optimization,up-scaling,and its partial characterization

Phytase production by Aspergillus niger NCIM 563 was optimized by using wheat bran in solid state fermentation (SSF). An integrated statistical optimization approach involving the combination of Placket–Burman design (PBD) and Box–Behnken design (BBD) was employed. PBD was used to evaluate the effect of 11 variables related to phytase production, and five statistically significant variables, namely, glucose, dextrin, NaNO₃, distilled water, and MgSO₄·7H₂O, were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a BBD. Phytase production improved from 50 IU/g dry moldy bran (DMB) to 154 IU/g DMB indicating 3.08-fold increase after optimization. A simultaneous reduction in fermentation time from 7 to 4 days shows a high productivity of 38,500 IU/kg/day. Scaling up the process in trays gave reproducible phytase production overcoming industrial constraints of practicability and economics. The culture extract also had 133.2, 41.58, and 310.34 IU/g DMB of xylanase, cellulase, and amylase activities, respectively. The partially purified phytase was optimally active at 55°C and pH 6.0. The enzyme retained ca. 75% activity over a wide pH range 2.0–9.5. It also released more inorganic phosphorus from soybean meal in a broad pH range from 2.5 to 6.5 under emulated gastric conditions. Molecular weight of phytase on Sephacryl S-200 was approximately 87 kDa. The K _m and V _max observed were 0.156 mM and 220 μm/min/mg. The SSF phytase from A. niger NCIM 563 offers an economical production capability and its wide pH stability shows its suitability for use in poultry feed.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

Enhanced production of poly (γ-glutamic acid) from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324 in solid state fermentation

This work reports on the optimization of PGA production by Bacillus licheniformis NCIM 2324 in solid state fermentation (SSF). In the first step, the one factor-at-a-time method was used to investigate the effect of solid substrates, initial moisture content, pH, and additional carbon and nitrogen source on PGA production; subsequently, response surface methodology (RSM) was used to establish the optimum concentrations of the key nutrients for higher PGA production. In the second step, the effects of amino acids and TCA cycle intermediates on the production of PGA were studied. The final optimized medium gave a maximum yield of 98.64 ± 1.61 mg gds⁻¹ of PGA, which is significantly higher than that reported in the literature.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Production of an Antifungal Chitinase from Enterobacter sp. NRG4 and its Application in Protoplast Production

Summary In this study flake chitin, crab shell chitin, mushroom stalk, fungal cell wall, wheat bran and rice bran were used as substrate for chitinase production by Enterobacter sp. NRG4 under submerged and solid state fermentation (SSF) conditions. Enterobacter sp. NRG4 produced 72 and 49.7 U/ml of chitinase in presence of cell walls of Candida albicans and Fusarium moniliforme in submerged fermentation. Under SSF, maximum chitinase production was 965 U/g solid substrate with flake chitin and wheat bran (1:3 ratio) at 75% moisture level after 144 h. The purified chitinase inhibited hyphal extension of Fusarium moniliforme, Aspergillus niger, Mucor rouxi and Rhizopus nigricans. The chitinase was effective in release of protoplasts from Trichoderma ressei, Pleurotus florida, Agaricus bisporus and Aspergillus niger. Protoplasts yield was maximum with 60 mg of 24 h old fungal mycelium incubated with 60 U of chitinase and 60 U of cellulase.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Chitinase Production in Solid-State Fermentation by Enterobacter sp. NRG4 Using Statistical Experimental Design

The optimization of nutrient levels for chitinase production by Enterobacter sp. NRG4 in solid-state fermentation conditions (SSF) was carried out using response surface methodology (RSM) based on central composite design (CCD). The design was employed by selecting wheat bran-to-flake chitin ratio, moisture level, inoculum size, and incubation time as model factors. The results of first-order factorial design experiments showed that all four independent variables have significant effects on chitinase production. The optimum concentrations for chitinase production were wheat bran-to-flake chitin ratio, 1; moisture level, 80%; inoculum size, 2.6 mL; and incubation time, 168 h. Using this statistical optimization method, chitinase production was found to increase from 616 U · g⁻¹ dry weight of solid substrate to 1475 U · g⁻¹ dry weight of solid substrate.     

Molecular genetic evidence for the transfer of Oerskovia species into the genus Cellulomonas

A close genetic relationship among strains of Oerskovia turbata, O. xanthineolytica and various coryneforms is indicated by DNA-DNA reassociation studies. O. xanthineolytica shares high homology values (over 60%) with Cellulomonas cartae, Nocardia cellulans, Brevibacterium fermentans and Corynebacterium manihot. O. turbata and other cellulomonads show lower DNA homology values (20–25%) which are still high enough, however, to indicate a relationship at the genus level. Based on these data and supported by comparative analysis of the ribosomal 16 S RNA and the similarity of peptidoglycan types, the transfer of Oerskovia species into the genus Cellulomonas is justified.This paper is respectively dedicated to our teacher and mentor, Professor Dr. O. Kandler, on the occasion of his 60th birthday.     

Callus formation,plant regeneration,and transient expression in the halophyte sea aster (<Emphasis Type="Italic">Aster tripolium</Emphasis> L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l⁻¹ casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1 × 10⁵ protoplasts ml⁻¹, 25 μg batch⁻¹ of plasmid vector, and 30% polyethylene glycol 4,000.     

The growth form of the inducing microorganism and chitin addition affect mycolytic enzyme production byTrichoderma harzianum

The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×10⁸ ml^–1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.     

Short communication: Protoplast formation from the thermophilic fungus Malbranchea sulfurea,using the thermostable chitinase and laminarinase of Paecilomyces varioti

Two thermostable enzymes produced by the thermophilic fungus Paecilomyces varioti, a chitinase and laminarinase, were used to isolate protoplasts of a thermophilic fungus, Malbranchea sulfurea. The frequency of protoplast regeneration observed (35%) was considerably higher than that obtained using commercial lytic enzymes.     

Ethanol production from alkali-treated rice straw via simultaneous saccharification and fermentation using newly isolated thermotolerant <Emphasis Type="Italic">Pichia kudriavzevii</Emphasis> HOP-1

In this study, simultaneous saccharification and fermentation (SSF) was employed to produce ethanol from 1% sodium hydroxide-treated rice straw in a thermostatically controlled glass reactor using 20 FPU gds⁻¹ cellulase, 50 IU gds⁻¹ β-glucosidase, 15 IU gds⁻¹ pectinase and a newly isolated thermotolerant Pichia kudriavzevii HOP-1 strain. Scanning electron micrograph images showed that the size of the P. kudriavzevii cells ranged from 2.48 to 6.93 μm in diameter while the shape of the cells varied from oval, ellipsoidal to elongate. Pichia kudriavzevii cells showed extensive pseudohyphae formation after 5 days of growth and could assimilate sugars like glucose, sucrose, galactose, fructose, and mannose but the cells could not assimilate xylose, arabinose, cellobiose, raffinose, or trehalose. In addition, the yeast cells could tolerate up to 40% glucose and 5% NaCl concentrations but their growth was inhibited at 1% acetic acid and 0.01% cyclohexamide concentrations. Pichia kudriavzevii produced about 35 and 200% more ethanol than the conventional Saccharomyces cerevisiae cells at 40 and 45°C, respectively. About 94% glucan in alkali-treated rice straw was converted to glucose through enzymatic hydrolysis within 36 h. Ethanol concentration of 24.25 g l⁻¹ corresponding to 82% theoretical yield on glucan basis and ethanol productivity of 1.10 g l⁻¹ h⁻¹ achieved using P. kudriavzevii during SSF hold promise for scale-up studies. An insignificant amount of glycerol and no xylitol was produced during SSF. To the best of our knowledge, this is the first study reporting ethanol production from any lignocellulosic biomass using P. kudriavzevii.     

Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion

Conditions were optimized for rapid release and improved regeneration of protoplasts ofSaccharomyces cerevisiae NCIM 3458. Rapid protoplast release was also obtained with representatives of several other yeast genera under the modified conditions of treatment. The application of the procedure in construction of a highly flocculentSaccharomyces cerevisiae with a killer character is described. Fusion was effected between UV-killed protoplasts ofS. cerevisiae NCIM 3578 with a killer character and live protoplasts of the highly flocculentS. cerevisiae NCIM 3528 in the presence of polyethylene glycol (PEG) 6000. Fusants were selected using benomyl resistance as marker, the killer toxin producer rather than the highly flocculent yeast being resistant to the fungicide at a concentration of 100 g ml^–1. Fusants were also characterized by their DNA contents, capacity for ethanolic fermentation of molasses sugar and levels of invertase, alcohol dehydrogenase and pyruvate decarboxylase activities.     

Lignocellulose-degrading enzyme production by white-rot <Emphasis Type="Italic">Basidiomycetes</Emphasis> isolated from the forests of Georgia

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml⁻¹) and xylanase (195 U ml⁻¹) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l⁻¹), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570–620 U l⁻¹) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.     

Development of an optimal culture system for callogenesis of <Emphasis Type="Italic">Chrysanthemum indicum</Emphasis> protoplasts

This study is a comparison of four methods to induce calli formation in a protoplast culture of Chrysanthemum indicum. Culture in liquid medium (17.6 calli/10⁵ protoplasts) was preferable to culture in solid agarose beads, although the efficiency of the latter could be improved by layering them on glass beads (12.5 vs. 0.83 calli/10⁵ protoplasts). Culture of protoplasts on moistened filter paper was unsuccessful. In the liquid media, microcalli and calli were induced efficiently and easily after 6 weeks. These effects may be explained by reduced toxicity due to cell breakdown and improved aeration.     

Extraction,purification and characterization of an antioxidant from marine waste using protease and chitinase cocktail

Marine waste is a highly renewable resource for the recovery of several value added metabolites with prospective industrial applications. This study describes the production of enzymes on marine waste and their subsequent use for the extraction of antioxidants from marine waste. Microbispora sp. and Bacillus sp. were grown on colloidal chitin and marine waste for the production of chitinase and protease. Microbispora sp. could produce 10.2 U ml⁻¹ chitinase, whereas Bacillus sp. could produce 38 U ml⁻¹ chitinase and 3.39 U ml⁻¹ protease. The production of antioxidants was optimized using statistical designs and 6.6 units of 35 kDa chitinase from Microbispora sp., 16 units of 25 kDa chitinase from Bacillus sp., 2.3 units of protease, 1.5% marine waste and 36 h incubation gave maximum antioxidant activity. Nearly 5.0 mg of compound with antioxidant activity could be recovered per gram of marine waste. This compound was purified by HPLC and characterized by TLC, FT-IR and proton-NMR as N,N′-diacetylchitobiose. It exhibited 53% superoxide radical scavenging activity, 57% hydroxyl radical scavenging activity and 28% lipid peroxidation inhibition activity. Scale up of the extraction of antioxidant from marine waste and its pharmacological studies can extend its use in medicine.     

Secretory production of cell wall components by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protoplasts in static liquid culture

When protoplasts of Saccharomyces cerevisiae T7 and IFO 0309 are cultured in a static liquid culture at 2.5 × 10⁶ protoplasts/ml, cell wall regeneration does not occur and cell wall components (CWC) are released into the culture broth. By using a specialized fluorometer, the concentrations of CWC could be measured on the basis of the fluorescence intensity of the CWC after staining with Fluostain I. The inoculum concentration, pH, and osmotic pressure of the medium were important factors for the production of CWC in culture. Under optimal culture conditions, S. cerevisiae T7 protoplasts produced 0.91 mg/ml CWC after 24 h. The CWC induced the tumor necrosis factor-α production about 1.3 times higher than that of the commercially available β-1,3/1,6-glucan from baker’s yeast cells.