Jyoti Vakhlu

Yeast lipases: enzyme purification, biochemical properties and gene cloning

Lipases are placed only after proteases and carbohydrases in world enzyme market and share about 5% of enzyme market. They occur in plants, animals and microorganisms and are accordingly classified as plant, animal and microbial lipases. Wherever they exist, they function to catalyze hydrolysis of triglycerides to glycerol and fatty acid. Like carbohydrases and proteases, lipases of microbial origin enjoy greater industrial importance as they are more stable (compared to plant and animal lipases) and can be obtained in bulk at low cost. Majority of yeast lipases are extracelluar, monomericglycoproteins with molecular weight ranging between ~33 to ~65 kD. More than 50% reported lipases producing yeast, produce it in the forms of various isozymes. These lipase isozymes are in turn produced by various lipase encoding genes. Among many lipase producing yeasts Candida rugosa is most frequently used yeast as the source of lipase commercially. This review is aimed at compiling the information on properties of various yeast lipases and genes encoding them.

High Throughput Sequencing: An Overview of Sequencing Chemistry

In the present century sequencing is to the DNA science, what gel electrophoresis was to it in the last century. From 1977 to 2016 three generation of the sequencing technologies of various types have been developed. Second and third generation sequencing technologies referred commonly to as next generation sequencing technology, has evolved significantly with increase in sequencing speed, decrease in sequencing cost, since its inception in 2004. GS FLX by 454 Life Sciences/Roche diagnostics, Genome Analyzer, HiSeq, MiSeq and NextSeq by Illumina, Inc., SOLiD by ABI, Ion Torrent by Life Technologies are various type of the sequencing platforms available for second generation sequencing. The platforms available for the third generation sequencing are Helicos™ Genetic Analysis System by SeqLL, LLC, SMRT Sequencing by Pacific Biosciences, Nanopore sequencing by Oxford Nanopore’s, Complete Genomics by Beijing Genomics Institute and GnuBIO by BioRad, to name few. The present article is an overview of the principle and the sequencing chemistry of these high throughput sequencing technologies along with brief comparison of various types of sequencing platforms available.

Metagenomics: Future of microbial gene mining

Modern biotechnology has a steadily increasing demand for novel genes for application in various industrial processes and development of genetically modified organisms. Identification, isolation and cloning for novel genes at a reasonable pace is the main driving force behind the development of unprecedented experimental approaches. Metagenomics is one such novel approach for engendering novel genes. Metagenomics of complex microbial communities (both cultivable and uncultivable) is a rich source of novel genes for biotechnological purposes. The contributions made by metagenomics to the already existing repository of prokaryotic genes is quite impressive but nevertheless, this technique is still in its infancy. In the present review we have drawn comparison between routine cloning techniques and metagenomic approach for harvesting novel microbial genes and described various methods to reach down to the specific genes in the metagenome. Accomplishments made thus far, limitations and future prospects of this resourceful technique are discussed.

Thermo-alkaliphilic halotolerant detergent compatible protease(s) of Bacillus tequilensis MTCC 9585

An extra-cellular thermo-alkaliphillic protease producing Bacillus was isolated from the soil and identified to be Bacillus tequilensis MTCC 9585 by microscopic, colony morphology, biochemical and 16S ribotyping. B. tequilensis MTCC 9585 produces protease up to 21 h of growth but interestingly 90% of the protease production occurred, just after 6 h of growth. The organism grew as well as and produced enzyme at wide pH (5 to 12) and temperature range (4, 25, 37 and 50°C), though optimum temperature and pH for the growth of the Bacillus were 37°C and pH 7.0. Optimum pH for enzyme activity coincided with optimum pH for enzyme production at pH 10. Optimum temperature for enzyme activity was 60°C and the enzyme stayed stable over the period of 270 days (9 months) at 10°C. Metal ions like Ca 2+ , Mg 2+ , K + increased the enzyme activity whereas Cu 2+ , Zn 2+ inhibit the activity slightly. Wash performance and stain removal efficiency increased when partially purified enzyme was used in conjunction with selected detergent. B. tequilensis can be a potential candidate for use in detergent industry because: of couple of reasons such as (i) 90% of the protease is produced only after 6 h of growth (economically viable), (ii) it’s activity in wide pH and temperature range (iii) it’s stability over the period of 9 months at 10°C indicating good shelf life and (iv) detergent compatibility.

Cloning and characterization of thermo-alkalistable and surfactant stable endoglucanase from Puga hot spring metagenome of Ladakh (J&K)

A thermo-alkalistable and surfactant stable endoglucanase (PHS) gene consisting of 554 amino acids was identified from metagenomic library of Puga hot spring using functional screening. PHS gene was overexpressed and purified to homogeneity using affinity chromatography The purified PHS protein presented a single band of 60kDa on the SDS-PAGE gel and zymogram. The recombinant PHS exhibited activity over a broad range of pH and temperature with optima at pH 8.0 and 65°C, respectively and having optimum stability at 60°C and pH 8.0, respectively. The recombinant PHS showed highest substrate specificity using CMC (218.4U/mg) as compared with Barley β-glucan (89.2U/mg) and Avicel (0.8U/mg). The Km and Vmax of recombinant PHS for CMC were 3.85mg/ml and 370.37μmolmin-1mg-1, respectively. The activity of the recombinant PHS was enhanced by treatment with 10mM non-ionic detergents such as Tween 20, Tween 40, Tween 80, Triton X- 100 and PEG and was inhibited by CTAB, SDS. Its functionality was stable in the presence of Fe3+ but inhibited by Cu2+, Hg2+, Mn2+ and Zn2+. These properties make PHS endoglucanase a potential candidate for use in laundry, textile,paper and pulp industries.

Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh, India to Explore Bacterial Diversity

ABSTRACT Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.

Comparative Metagenomics Reveal Phylum Level Temporal and Spatial Changes in Mycobiome of Belowground Parts of Crocus sativus

Plant-fungal associations have been explored by routine cultivation based approaches and cultivation based approaches cannot catalogue more than 5% of fungal diversity associated with any niche. In the present study, an attempt has been made to catalogue fungal diversity associated with belowground parts i.e. rhizosphere and cormosphere, of Crocus sativus (an economically important herb) during two growth stages, using cultivation independent ITS gene targeted approach, taking bulk soil as reference. The 454 pyrosequencing sequence data analysis suggests that the fungal diversity was niche and growth stage specific. Fungi diversity, in the present case, was not only different between the two organs (roots and corm) but the dominance pattern varies between the cormosphere during two growth stages. Zygomycota was dominant fungal phylum in the rhizosphere whereas Basidiomycota was dominant in cormosphere during flowering stage. However in cormosphere though Basidiomycota was dominant phylum during flowering stage but Zygomycota was dominant during dormant stage. Interestingly, in cormosphere, the phyla which was dominant at dormant stage was rare at flowering stage and vice-versa (Basidiomycota: Flowering = 93.2% Dormant = 0.05% and Zygomycota: Flowering = 0.8% Dormant = 99.7%). At genus level, Rhizopus was dominant in dormant stage but was rare in flowering stage (Rhizopus: Dormant = 99.7% Flowering = 0.55%). This dynamics is not followed by the bulk soil fungi which was dominated by Ascomycota during both stages under study. The genus Fusarium, whose species F. oxysporum causes corm rot in C. sativus, was present during both stages with slightly higher abundance in roots. Interestingly, the abundance of Rhizopus varied a great deal in two stages in cormosphere but the abundance of Fusarium was comparable in two growth stages (Bulk soil Flowering = 0.05%, Rhizosphere Flowering = 1.4%, Cormosphere Flowering = 0.06%, Bulk soil Dormant = 2.47% and cormosphere dormant = 0.05%). This is the first report on the fungal diversity associated with the root of Crocus sativus and first report on the fungi associated with corm of any plant with the temporal and spatial variation in the fungal community structure.

Draft Genome Sequence of Plant Growth-Promoting Bacillus amyloliquefaciens Strain W2 Associated with Crocus sativus (Saffron)

ABSTRACT Bacillus sp. strain W2 is a plant growth-promoting rhizobacterium isolated from saffron fields of Kashmir, India. Here, we report the draft genome sequence (3.9 Mb) of Bacillus amyloliquefaciens strain W2 having 65 contigs (3, 997, 511 bp), 4,163 coding sequences, and an average 46.45% GC content. Despite the 99% identity of the 16S rRNA gene with that of Bacillus amyloliquefaciens subsp. plantarum FZB42, the genome comparison revealed that only 48.7% of the W2 genome has homology with that of FZB42.

Metagenomics: A Relief Road to Novel Microbial Genes and Genomes

A huge quantum of the genetic and metabolic diversity of the biosphere is locked up within the microbial communities present in and on earth, as majority of earth’s biomass comprises microbes. Ironically we cannot access more than 1% of this bioresource through routine culturing techniques as we have underestimated the power of microbes to resist culturing. This 1% of the microbial diversity accessed so far contributes to more than 80% of the industrial biotechnology at present and what miracles rest of the 99% can perform is anybody’s guess. Metagenomics (also known as e-genomics, community genomics and environmental genomics) is clutch of molecular biology techniques used to hunt the culture resisting, yet- to- be cultured microbes. Metagenomics is a fruit born of the wedlock between modern molecular techniques and the idea, that microbial diversity can be analyzed by direct DNA isolation from environmental samples (metagenomic DNA), it’s cloning, screening and sequencing. It would not be farfetched to conclude that after invention of microscope and culturing techniques, metagenomics is third big revolution in microbiology. Metagenomic revolution would not have been possible without the availability of low cost DNA sequencing services, bioinformatics tools and high through put screening techniques. This chapter will deal with the techniques used in metagenomics and achievements made so far along with future applications and challenges.

Phylogenetic diversity and metabolic potential of microbiome of natural healing clay from Chamliyal (J&K)

Clay therapy for skin disease treatment is an ancient practice popular worldwide as a cheap alternative to pharmaceutical products. Effectiveness of clay against skin problems has been linked to its mineral composition and to microbial activity. The clay–water paste of a holy shrine Chamliyal in the Jammu region of J&K, India is used as an ointment to treat different skin disorders particularly psoriasis. Using the 16 SrDNA amplicon pyrosequencing and whole-metagenome direct shotgun Illumina sequencing, microbial phylogeny and potential metabolic functions were catalogued for Chamliyal’s clay. Microbial diversity profile of the Chamliyal’s clay is similar to other medicinal clays, particularly Dead Sea; there is some uniqueness as well. Although Proteobacteria, Actinomycetes and Firmicutes are common inhabitants of all the clay types, sulphur- and iron-reducing bacteria like Deferribacterales are particular to clays used for skin healing. In the present study it is proposed that healing properties of clay may be due to the microbes and microbial genes associated with metabolism of minerals like iron and sulphur, that lead to mineral acquisition in the Chamliyal’s clay.