Sathyanarayana N. Gummadi

Purification and biochemical properties of microbial pectinases—a review

Abstract Pectinases are a complex group of enzymes that degrade various pectic substances present in plant tissues. Pectinases have potential applications in fruit, paper and textile industries. Apart from these industrial applications, these enzymes possess biological importance in protoplast fusion technology and plant pathology. Since applications of pectinases in various fields are widening, it is important to understand the nature and properties of these enzymes for efficient and effective usage. For the past few years, vigorous research has been carried out on isolation and characterization of pectinases. New affinity matrices with improved characteristics and affinity-precipitation techniques have been developed for purification of pectinases. Recently much attention has been focused on chemical modification of pectinases and their catalytic performance by various researchers. These studies are helpful in determining key amino acid residues responsible for substrate binding, catalytic action, and physico-chemical environmental conditions for maximum hydrolysis. This short review highlights progress on purification and understanding the biochemical aspects of microbial pectinases.

Catabolic pathways and biotechnological applications of microbial caffeine degradation

Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonasputida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.

Microbial pectic transeliminases

Pectic transeliminases, also known as pectic lyases or pectinases, are involved in the degradation of pectic substances. They have a wide range of applications in food and textile processing. Although Aspergillus and Penicillium spp. produce pectin lyases, bacteria are the major producers of polygalacturonate lyase. The yields of pectic transeliminases are less than other pectinases. Since new applications for pectic transeliminases are emerging, an improved process for the production of these enzymes is necessary.

What is the role of thermodynamics on protein stability

The most challenging and emerging field of biotechnology is the tailoring of proteins to attain the desired characteristic properties. In order to increase the stability of proteins and to study the function of proteins, the mechanism by which proteins fold and unfold should be known. It has been debated for a long time how exactly the linear form of a protein is converted into a stable 3-dimensional structure. The literature showed that many theories support the fact that protein folding is a thermodynamically controlled process. It is also possible to predict the mechanism of protein deactivation and stability to an extent from thermodynamic studies. This article reviewed various theories that have been proposed to explain the process of protein folding after its biosynthesis in ribosomes. The theories of the determination of the thermodynamic properties and the interpretation of thermodynamic data of protein stability are also discussed in this article.

Physiology, biochemistry and possible applications of microbial caffeine degradation

Caffeine, a purine alkaloid is a constituent of widely consumed beverages. The scientific evidence which has proved the harm of this alkaloid has paved the way for innumerable research in the area of caffeine degradation. In addition to this, the fact that the by-products of the coffee and tea industry pollute the environment has called for the need of decaffeinating coffee and tea industry’s by-products. Though physical and chemical methods for decaffeination are available, the lack of specificity for removal of caffeine in these techniques and their non-eco-friendly nature has opened the area of microbial and enzymatic degradation of caffeine. Another important application of microbial caffeine degradation apart from its advantages like specificity, eco-friendliness and cost-effectiveness is the fact that this process will enable the production of industrially and medically useful components of the caffeine degradation pathway like theobromine and theophylline. This is a comprehensive review which mainly focuses on caffeine degradation, large-scale degradation of the same and its applications in the industrial world.

Production of extracellular water insoluble β-1,3-glucan (curdlan) fromBacillus sp. SNC07

Abstractβ-1,3-Glucan (curdlan) is a water-insoluble polysaccharide composed exclusively of β-1,3 linked glucose residues. Extracellular curdlan was mostly synthesized byAgrobacterium species andAlcaligenes faecalis under nitrogen-limiting conditions. In this study, we screened the microorganisms capable of producing extracellular curdlan from soil samples. For the first time, we reported Gram-positive bacteriumBacillus sp. SNC 107 capable of producing extracellular curdlan in appreciable amounts. The effect of different carbon sources on curdlan production was studied and found that the yield of curdlan was more when glucose was used as carbon source. It was also found that maximum production was achieved when the initial concentration of ammonium and phosphate in the medium was 0.5 and 1.9 g/L respectively. In this study the curdlan production was increased from 3 to 7 g/L in shake flask cultures.

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

BACKGROUND Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown. METHODS In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. RESULTS The results in this study show that (i) affinities of the peptides are in the order hPLSCR1>hPLSCR3>hPLSCR2>hPLSCR4 for Ca2+ and in the order hPLSCR1>hPLSCR2>hPLSCR3>hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. CONCLUSIONS Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif. GENERAL SIGNIFICANCE Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.

Production and downstream processing of (1→3)-β-D-glucan from mutant strain of Agrobacterium sp. ATCC 31750

We isolated a mutant that produced higher levels of curdlan than the wild strain Agrobacterium sp. ATCC 31750 by chemical mutagenesis using N-methyl-N-nitro-nitrosoguanidine. The mutant strain produced 66 g/L of curdlan in 120 h with a yield of (0.88) while, the wild strain produced 41 g/L in 120 h with a yield of (0.62) in a stirred bioreactor. The mutant could not produce curdlan when the pH was shifted from 7.0 to 5.5 after nitrogen depletion as followed for wild strain. In contrast, pH optimum for cell growth and curdlan production for mutant was found to be 7.0. We optimized the downstream processing of curdlan by varying different volumes of NaOH and HCl for extraction and precipitation of curdlan. The molecular weight of the purified curdlan from the wild and mutant strain was 6.6 × 105 Da and 5.8 × 105 Da respectively. The monosaccharide analyses confirm that curdlan from both wild and mutant strain contains only glucose units. From the NMR and FTIR data, it has been confirmed that curdlan was exclusively composed of β (1 → 3)-D-glucan residues.

Structural and Biochemical Properties of Pectinases

Pectin and other pectic substances are complex polysaccharides, which contribute firmness and structure to plant tissues as a part of the middle lamella. The basic unit in pectic substances is galacturonan ( -D-galacturonic acid). Pectic substances are classified into two types; homogalacturonan and heterogalacturonan (rhamnogalacturonan). In homogalacturonan, the main polymer chain consists of -D-galacturonate units linked by 1 → 4 glycosidic bonds, whereas in rhamnogalacturonan, the primary chain consist of 1→ 4 linked -D-glacturonates and with about 2–4% L-rhamnose units that are 1→ 2 and 1→ 4 linked to D-galacturonate units (Whitaker, 1991). The side chains of rhamnogalacturonans usually consist of L-arabinose or D-galacturonic acid units. In plant tissues, about 60–70% of the galacturonate units are esterified with methanol and occasionally with ethanol. Based on the degree of esterification, pectic substances are classified into protopectin, pectinic acid, pectin and polygalacturonic acid (Table 1). Molecular size, degree of esterification and weight distribution of polygalacturonic acid residues are important factors that contribute to heterogeneity in pectic substances. Relative molecular masses of pectic substances isolated from various sources such as citrus fruits, apple and plums, range from 25 to 350 kDa. Pectinases are a complex and diverse group of enzymes involved in the degradation of pectic substances. The diversity of forms of pectic substances in plant cells probably accounts for the existence of various forms of these enzymes. Pectinases are classified depending on their substrate and mode of enzymatic reaction (Fig. 1). Pectinases act as carbon recycling agents in nature by degrading pectic substances to saturated and unsaturated galacturonans, which are further catabolized

The single C‐terminal helix of human phospholipid scramblase 1 is required for membrane insertion and scrambling activity

Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP‐independent class of phospholipid translocators which possess a single EF‐hand‐like Ca2+‐binding motif and also a C‐terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C‐terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for ∆CTH‐hPLSCR1 (Ka = 115 μm) compared with hPLSCR1 (Ka = 249 μm). Far UV‐CD studies revealed that the CTH peptide adopts α‐helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide‐quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1.