Description of lipase producing novel yeast species Debaryomyces apis f.a., sp. nov. and a modified pH indicator dye-based method for the screening of lipase producing microorganisms.

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23HT showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095T, 7.8% from Debaryomyces udenii JCM 7855T, and Debaryomyces coudertii JCM 2387T in the D1/D2 domain. In the ITS region sequences, strain CIG-23HT showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095T and Debaryomyces coudertii JCM 2387T. In 18S rRNA gene sequence, the strain CIG-23HT showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23HT can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23HT is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297T, and the isotypes are MTCC 12914T and KCTC 37024T. The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Whole-Genome Sequence Data Analysis of Anoxybacillus kamchatkensis NASTPD13 Isolated from Hot Spring of Myagdi, Nepal.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

Comparative genomics of Sporothrix species and identification of putative pathogenic-gene determinants.

Aim: To understand the phylogenomics, pathogenic/virulence-associated genes and genomic evolution of pathogenic Sporothrix species. Materials & methods: We performed in silico comparative genome analysis of Sporothrix species using ab initio tools and in-house scripts. We predicted genes and repeats, compared genomes based on synteny, identified orthologous clusters, assessed genes family expansion/contraction, predicted secretory proteins and finally searched for similar sequences from various databases. Results: The phylogenomics revealed that Sporothrix species are closely related to Ophiostoma species. The gene family evolutionary analysis revealed the expansion of genes related to virulence (CFEM domain, iron acquisition genes, lysin motif domain), stress response (Su[var]3-9, Enhancer-of-zeste and Trithorax domain and Domain of unknown function 1996), proteases (aspartic protease, x-pro dipeptidyl-peptidase), cell wall composition associated genes (chitin deacetylase, chitinase) and transporters (major facilitator superfamily transporter, oligo-peptide transporter family) in Sporothrix species. Conclusion: The present study documents the putative pathogenic/virulence-associated genes in the Sporothrix species.

Understanding the Effect of Impeller Configurations on Pullulan Production by Aureobasidium Pullulans RBF 4A3.

Mass transfer is one of the most important factors involved in viscous fermentation processes, like production of pullulan. Impellers play a crucial role in maintaining homogeneity and better mass transfer conditions during the fermentation process. The present study attempted to evaluate the efficiency of impellers with diverse configurations during pullulan fermentation. Initially, the mass transfer coefficients of 10 selected impellers were evaluated in an aqueous system. Among these, three impellers, namely, single helical ribbon, Rushton turbine, and Smith turbine impellers, were found to be more efficient and were further employed in the pullulan fermentation process. The results suggested that the single helical ribbon impeller was able to provide 24% higher pullulan production as compared to the Rushton turbine and Smith turbine impellers. The single helical ribbon was able to maintain the critical demand of dissolved oxygen in fermentation broth. Therefore, it may be commented that the single helical ribbon impeller configuration is suitable for higher production of pullulan during the fermentation process.

Biochemical Characterization of Kluyveromyces lactis Adenine Deaminase and Guanine Deaminase and Their Potential Application in Lowering Purine Content in Beer.

Excess amounts of uric acid in humans leads to hyperuricemia, which is a biochemical precursor of gout and is also associated with various other disorders. Gout is termed as crystallization of uric acid, predominantly within joints. The burden of hyperuricemia and gout has increased worldwide due to lifestyle changes, obesity, and consumption of purine-rich foods, fructose-containing drinks, and alcoholic beverages. Some of the therapies available to cure gout are associated with unwanted side-effects and antigenicity. We propose an attractive and safe strategy to reduce purine content in beverages using enzymatic application of purine degrading enzymes such as adenine deaminase (ADA) and guanine deaminase (GDA) that convert adenine and guanine into hypoxanthine and xanthine, respectively. We cloned, expressed, purified, and biochemically characterized both adenine deaminase (ADA) and guanine deaminase (GDA) enzymes that play important roles in the purine degradation pathway of Kluyveromyces lactis, and demonstrate their application in lowering purine content in a beverage. The popular beverage beer has been selected as an experimental sample as it confers higher risks of hyperuricemia and gout. Quantification of purine content in 16 different beers from the Indian market showed varying concentrations of different purines. Enzymatic treatment of beer samples with ADA and GDA showed a reduction of adenine and guanine content, respectively. These enzymes in combination with other purine degrading enzymes showed marked reduction in purine content in beer samples. Both enzymes can work at 5.0-8.0 pH range and retain >50% activity at 40°C, making them good candidates for industrial applications.

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13.

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6-9) and temperature (30-65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 µM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages.

Malassezia arunalokei sp. nov., a Novel Yeast Species Isolated from Seborrheic Dermatitis Patients and Healthy Individuals from India.

The majority of species within the genus Malassezia are lipophilic yeasts that colonize the skin of warm-blooded animals. Two species, Malassezia globosa and Malassezia restricta, are implicated in the causation of seborrheic dermatitis/dandruff (SD/D). During our survey of SD/D cases, we isolated several species of Malassezia and noticed vast variations within a few lipid-dependent species. Variations observed in the phenotypic characteristics (colony morphology, absence of catalase activity, growth at 37°C, and precipitation surrounding wells containing Tween 20 or Cremophor EL) suggested the possible presence of a novel species. Sequence divergence observed in the internal transcribed spacer (ITS) region, the D1/D2 domain, and the intergenic spacer 1 (IGS1) region of rDNA and the TEF1 gene, PCR-restriction fragment length polymorphism (RFLP) analysis of the ITS2 region, and fluorescent amplified fragment length polymorphism analysis support the existence of a novel species. Based on phenotypic and molecular characterization of these strains, we propose a new species, namely, M. arunalokei sp. nov., and we designate NCCPF 127130 (= MTCC 12054 = CBS 13387) as the type strain.

Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Ålesund, Arctic.

A psychrophilic yeast species was isolated from glacier cryoconite holes of Svalbard. Nucleotide sequences of the strains were studied using D1/D2 domain, ITS region and partial sequences of mitochondrial cytochrome b gene. The strains belonged to a clade of psychrophilic yeasts, but showed marked differences from related species in the D1/D2 domain and biochemical characters. Effects of temperature, salt and media on growth of the cultures were also studied. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activities was carried out. The strains expressed high amylase and lipase activities. Freeze tolerance ability of the isolates indicated the formation of unique hexagonal ice crystal structures due to presence of 'antifreeze proteins' (AFPs). FAME analysis of cultures showed a unique trend of increase in unsaturated fatty acids with decrease in temperature. The major fatty acids recorded were oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, myristic acid and pentadecanoic acid. Based on sequence data and, physiological and morphological properties of the strains, we propose a novel species, Rhodotorula svalbardensis and designate strains MLB-I (CCP-II) and CRY-YB-1 (CBS 12863, JCM 19699, JCM 19700, MTCC 10952) as its type strains (Etymology: sval.bar.den'sis. N.L. fem. adj. svalbardensis pertaining to Svalbard).

Development of suitable solvent system for downstream processing of biopolymer pullulan using response surface methodology.

Downstream processing is an important aspect of all biotechnological processes and has significant implications on quality and yield of the final product. Several solvents were examined for their efficacy on pullulan precipitation from fermentation broth. Interactions among four selected solvents and their effect on pullulan yield were studied using response surface methodology. A polynomial model was developed using D-optimal design and three contour plots were generated by performing 20 different experiments and the model was validated by performing optimization experiments. The results indicated that lower concentration of ethanol in combination with the other three solvents has resulted in higher yield of polymer from fermentation broth and the optimized solvent system was able to recover 1.44 times more pullulan as compared to the conventional ethanolic precipitation method. These observations may help in enhancing efficiency of pullulan recovery from fermentation broth and also result in reduced cost of production for the final product.

The role of culture collections as an interface between providers and users: the example of yeasts.

The importance and species diversity of yeasts in food production are described, including a listing of agricultural applications. Two yeast species were selected for case studies on distribution patterns from microbial culture collections: the high representation of Saccharomyces cerevisiae in culture collections enabled global comparison, while Dekkera bruxellensis deposits and distributions were analyzed from the perspective of a single culture collection. In conclusion, culture collections need to cover temporal gaps between deposit and application of strains. The further development of culture collections in countries of high but underexplored species diversity should facilitate the conservation and management of microbial resources.

Candida ruelliae sp. nov., a novel yeast species isolated from flowers of Ruellia sp. (Acanthaceae).

Two novel yeast strains designated as 16Q1 and 16Q3 were isolated from flowers of the Ruellia species of the Acanthaceae family. The D1/D2 domain and ITS sequences of these two strains were identical. Sequence analysis of the D1/D2 domain of large-subunit rRNA gene indicated their relationship to species of the Candida haemulonii cluster. However, they differ from C. haemulonii by 14% nucleotide sequence divergence, from Candida pseudohaemulonii by 16.1% and from C. haemulonii type II by 16.5%. These strains also differ in 18 physiological tests from the type strain of C. haemulonii, and 12 and 16 tests, respectively, from C. pseudohaemulonii and C. haemulonii type II. They also differ from C. haemulonii and other related species by more than 13% sequence divergence in the internal transcribed spacer region. In the SSU rRNA gene sequences, strain 16Q1 differs by 1.7% nucleotide divergence from C. haemulonii. Sporulation was not observed in pure or mixed cultures on several media examined. All these data support the assignment of these strains to a novel species; we have named them as Candida ruelliae sp. nov., and designate strain 16Q1(T)=MTCC 7739(T)=CBS10815(T) as type strain of the novel species.

Debaryomyces singareniensis sp. nov., a novel yeast species isolated from a coal mine soil in India.

Three strains (AP19, AP19-4 and AP19-6) of a novel yeast species were isolated from soil from the Singareni coal mines, Andhra Pradesh, India. They were morphologically, physiologically and phylogenetically identical and produced one to four spherical ascospores per ascus. Phylogenetic analysis using the D1/D2 variable domain of the large-subunit rRNA gene indicated that the closest relative of these strains is Debaryomyces etchellsii (2.6% divergence). Other species related to these strains are D. mycophilus (5.1% divergence) and species of the D. hansenii cluster (4.9-5.6% divergence). The novel species differs by 20 and 15 physiological tests from D. etchellsii and D. mycophilus, respectively. Phylogenetic analysis of the internal transcribed spacer (ITS) region also indicated that strains of the new species are related to D. etchellsii (7.7% divergence), followed by species of the D. hansenii cluster (9-10% divergence). In the small-subunit rRNA gene sequences, they differed from D. etchellsii by seven substitutions and one insertion or deletion of a base in a sequence (indel) and from D. mycophilus by 17 substitutions and 1 indel. The physiological, biochemical and molecular data suggest that these strains belong to a novel species, for which we propose the name Debaryomyces singareniensis sp. nov. The type strain of AP19(T) (=MTCC 7061(T)=CBS 10405(T)). The Mycobank number of the new species is MB510046.