Bioprospecting Psychrotrophic Bacteria for Serine-Type Proteases from the Cold Areas of Western Himalayas.

The aim of present study was to analyze the prevalence of protease diversity among psychrotrophic bacteria in Lahaul and Spiti of the Western Himalayas. A total of 459 bacteria were screened and protease activity was observed in 150 isolates at 5 °C. Furthermore, 55 isolates showed protease activity up to pH 10 at 5 °C. Based on the hydrolytic zone, 22 isolates were selected for protease quantification. The protease activity varied from 58-377 U mL-1 at 10 °C, 49-396 U mL-1 at 28 °C and 31-407 U mL-1 at 37 °C. Similarly, protease activity ranged from 36-353 U mL-1 at pH 7, 40-306 U mL-1 at pH 9 and 33-304 U mL-1 at pH 10. The isolates were identified based on 16S rRNA gene sequencing and showed phylogenetic relationship to Arthrobacter belonging to the class Actinobacteria, Bacillus, Exiguobacterium, Paenibacillus, and Planomicrobium to Bacilli, and Pseudomonas, Serratia, and Stenotrophomonas to Gammaproteobacteria. Zymogram analysis revealed variations in protease isoforms ranging from 20 to 250 kDa which were strongly inhibited in the presence of phenylmethylsulfonyl fluoride, thus indicated serine-type nature. The extensive number of serine proteases among these bacteria was confirmed by annotating genomes of the reported genera for prevalence of protease isoforms. The properties of proteases including low-temperature activity with alkaline stability and detergent compatibility suggested their suitability as bio-additives in laundry.

Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12).

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323bp ORF encoding 441 amino acids protein with molecular weight 47.2kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1-21 amino acids, pre-peptide 22-143 amino acids, peptidase S8 domain 144-434 amino acids, and pro-peptide 435-441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed ∼50kDa and ∼40kDa bands, respectively. The pre-propeptide ∼11kDa removed from Alp1 resulted in mature protein of ∼35kDa with 1738Umg-1 specific activity. The recombinant protease was optimally active at 40°C and pH 9, and stable over 10-70°C and 6-12pH. The activity at low-temperature and alkaline pH was supported by high R/(R+K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry.

Complete genome sequence of a low-temperature active and alkaline-stable endoglucanase-producing Paenibacillus sp. strain IHB B 3084 from the Indian Trans-Himalayas.

A genome of 5.88Mb with 46.83% G+C content is reported for an endoglucanase-producing bacterium Paenibacillus sp. strain IHB B 3084 isolated from the cold environments of the Indian Trans-Himalayas. The psychrotrophic bacterium produces low-temperature active and alkaline-stable endoglucanases of industrial importance. The genomic data has provided insight into genomic basis of cellulase production and survival of the bacterium in the cold environments.

Draft Genome Sequence of a Cellulase-Producing Psychrotrophic Paenibacillus Strain, IHB B 3415, Isolated from the Cold Environment of the Western Himalayas, India.

Paenibacillus sp. strain IHB B 3415 is a cellulase-producing psychrotrophic bacterium isolated from a soil sample from the cold deserts of Himachal Pradesh, India. Here, we report an 8.44-Mb assembly of its genome sequence with a G+C content of 50.77%. The data presented here will provide insights into the mechanisms of cellulose degradation at low temperature.

Draft Genome Sequence of Psychrotrophic Acinetobacter sp. Strain MN12 (MTCC 10786), Which Produces a Low-Temperature-Active and Alkaline-Stable Peptidase.

We report here the draft genome sequence of Acinetobacter sp. strain MN12 (MTCC 10786), which is a psychrotrophic bacterium that produces an extracellular low-temperature-active and alkaline-stable peptidase. The draft genome assembly of Acinetobacter sp. MN12 has a size of 4.31 Mbp, with a G+C content of 40.75%.

First draft genome sequence of a member of the genus planomicrobium, isolated from the chandra river, India.

We report the first draft genome sequence of a member of the genus Planomicrobium, isolated from a soil sample from the Chandra River, located in the cold deserts of Himachal Pradesh, India. The draft genome assembly for Planomicrobium glaciei strain CHR43 has a size of 3,900,800 bp with a G+C content of 46.97%.

Purification and Characterization of an Extracellular Low Temperature-Active and Alkaline Stable Peptidase from Psychrotrophic Acinetobacter sp. MN 12 MTCC (10786).

An extracellular low temperature-active alkaline stable peptidase from Acinetobacter sp. MN 12 was purified to homogeneity with a purification fold of 9.8. The enzyme exhibited specific activity of 6,540 U/mg protein, with an apparent molecular weight of 35 kDa. The purified enzyme was active over broad range of temperature from 4 to 60 °C with optimum activity at 40 °C. The enzyme retained more than 75 % of activity over a broad range of pH (7.0-11.0) with optimum activity at pH 9.0. The purified peptidase was strongly inhibited by phenylmethylsulfonyl fluoride, giving an indication of serine type. The K m and V max for casein and gelatin were 0.3529, 2.03 mg/ml and 294.11, 384.61 µg/ml/min respectively. The peptidase was compatible with surfactants, oxidizing agents and commercial detergents, and effectively removed dried blood stains on cotton fabrics at low temperature ranging from 15 to 35 °C.

Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth.

BACKGROUND: Guadua angustifolia Kunth is a very important bamboo species with significant utility in pharmaceutical, paper, charcoal, and construction industries. Microbial contamination is a major problem encountered during establishment of in vitro cultures of Guadua. OBJECTIVE: This study has been designed to analyze the identity of contaminating bacteria and to develop the strategy to eliminate them during micropropagation of Guadua. MATERIALS AND METHODS: We isolated and consequently analyzed partial sequence analysis of the 16S rRNA gene to identify two contaminating bacteria as (1) Pantoea agglomerans and (2) Pantoea ananatis. In addition, we also- performed antibiotic sensitivity testing on these bacterial isolates. RESULTS: We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria. We grew shoots on multiplication medium containing BAP (2 mg/l) and adenine sulfate (10 mg/l) supplemented with kanamycin (10 µg/ml) for 10 days and transferred them to fresh medium without antibiotics and found that bacterial growth was inhibited. Moreover, we observed intensive formation of high-quality shoots. Streptomycin sulfate also inhibited bacterial growth but at higher concentration. We also demonstrated that shoots grown in streptomycin sulfate tended to be shorter and had yellow leaves. CONCLUSION: Thus, we have developed a novel strategy to identify and inhibit intriguing microbial contaminations of (1) Pantoea agglomerans and (2) Pantoea ananatis during establishment of in vitro cultures of Guadua. This would improve in vitro establishment of an important bamboo, Guadua angustifolia Kunth for large scale propagation.

Microbial proteases: detection, production, and genetic improvement.

Microbial proteases are one of the important groups of industrially and commercially produced enzymes contributing approximately 2/3 of all enzyme sales. Though proteases are produced by many microorganisms, emphasis is on the microorganisms producing proteases with desired characters. As demand for novel proteases is increasing day by day the initial screening methods and assays for protease detection are of utmost importance. This review focuses attention on present status of knowledge on the various methods and protocols available for protease screening, detection, and quantification starting from plate assays to spectrophotometric, fluorometric, and nanoparticles based assays. The review will help in making strategies for exploitation of protease resources and improvement of enzymes to obtain more robust proteases.

Phylogenetic diversity of alkaline protease-producing psychrotrophic bacteria from glacier and cold environments of Lahaul and Spiti, India.

The diversity of proteolytic bacteria associated with a glacier and cold environment soils from three different locations in Lahaul and Spiti, India was investigated. Two hundred seventeen bacterial strains were isolated in pure culture. Subsequently these strains were screened for protease-production and one hundred nine showed protease production. From these protease producing psychrotrophic bacteria twenty showing high enzyme production at low temperature and alkaline pH were characterized and identified. The 16S rRNA phylogenetic analysis revealed that none of the strains showed 100% identity with the validly published species of various genera. Isolates belonged to three classes i.e. Actinobacteria, Gammaproteobacteria and Alphaproteobacteria, and were affiliated with the genera Acinetobacter, Arthrobacter, Mycoplana, Pseudomonas, Pseudoxanthomonas, Serratia and Stenotrophomonas. The optimal growth temperature ranged from 10 to 28 degrees C and interestingly, high levels of enzyme productions were measured at growth temperatures between 15 and 25 degrees C, for most of the isolates in plate assay. Most of the isolates were found to produce at least two other hydrolytic enzymes along with protease. The crude protease from one strain was active over broad range of temperature and pH with optima at 30 degrees C and 7.5, respectively. The protease activity was enhanced by Ca(2+), dithiothreitol and beta-mercaptoethanol. While Na(+), Hg(2+), Zn(2+), Mn(2+), phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid did not showed much effect on protease activity. The results enrich our knowledge on the psychrotrophic bacterial diversity and biogeographic distribution of enzyme producing bacteria in western Himalaya.

Proteases from psychrotrophs: an overview.

Proteases are hydrolytic enzymes which catalyze the total hydrolysis of proteins in to amino acids. Although proteolytic enzymes can be obtained from animals and plants but microorganisms are the preferred source for industrial applications in view of scientific and economical advantage. Among various groups of microbes, psychrotrophs are ideal candidates for enzymes production keeping in mind that enzymes active at low temperature and stable under alkaline condition, in presence of oxidants and detergents are in large demand as laundry additive. The proteases from psychrotrophs also find application in environmental bioremediation, food and molecular biology. During the previous two decades, proteases from psychrotrophs have received increased attention because of their wide range of applications, but the full potential of psychrotrophic proteases has not been exploited. This review focuses attention on the present status of knowledge on the production, optimization, molecular characteristics, applications, substrate specificity, and crystal structure of psychrotrophic proteases. The review will help in making strategies for exploitation of psychrotrophic protease resources and improvement of enzymes to obtain more robust proteases of industrial and biotechnological significance.

Plant growth-promoting and rhizosphere-competent Acinetobacter rhizosphaerae strain BIHB 723 from the cold deserts of the Himalayas.

A phosphate-solubilizing bacterial strain BIHB 723 isolated from the rhizosphere of Hippophae rhamnoides was identified as Acinetobacter rhizosphaerae on the basis of phenotypic characteristics, carbon source utilization pattern, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The strain exhibited the plant growth-promoting attributes of inorganic and organic phosphate solubilization, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, ammonia generation, and siderophore production. A significant increase in the growth of pea, chickpea, maize, and barley was recorded for inoculations under controlled conditions. Field testing with the pea also showed a significant increment in plant growth and yield. The rifampicin mutant of the bacterial strain effectively colonized the pea rhizosphere without adversely affecting the resident microbial populations.

A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine.

Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram's iodine instead of the reagents just mentioned. Gram's iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram's iodine for the detection of cellulase production by microorganisms using plate assay.

Isolation and identification of a psychrotrophic Acinetobacter sp. CR9 and characterization of its alkaline lipase.

Forty three psychrotrophic bacteria were isolated from soil samples collected from Chandra river in sub-alpine region of western Himalaya, India. Among these, 11 isolates were found positive for lipase production at low temperature. Of 11 isolates, CR9 produced largest zone of clearance on plate assay and was able to produce lipase under wide range of pH. The isolate CR9 was identified as Acinetobacter sp. based on morphological and physiochemical characterization and 16S rRNA gene sequencing analysis. According to 16S rRNA gene sequencing data the closest phylogenetic neighbor for strain CR9 was Acinetobacter lwoffii (98.9%). The partially purified lipase from strain CR9 exhibited maximum activity at temperature 40 degrees C and pH optima at 8.0. Cu(2+), Mo(2+), Mg(2+), Zn(2+), phenylmethanesulfonyl fluoride (PMSF), dithiothreitol (DTT) and beta-mercaptoethanol (2-ME) enhanced the enzyme activity, whereas Ca(2+) and ethylenediaminetetraacetic acid (EDTA) had inhibitory effect. Lipase hydrolyzed wide range of short chain fatty acid esters of p-nitrophenyl. The organism CR9 also hydrolyzed tributyrin, Tween 80, soybean oil, mustard oil and olive oil. The results highlight the relevance of unexplored microbes from cold environments of western Himalaya for the isolation of novel lipase producing bacteria.