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There is threefold reduction in water usage and less chromium discharge into the environment

In a jiffy: It takes just 10 minutes to open the fibres when the biocatalyst is used compared with traditional enzymes that take three-four hours. APDar Yasin

A novel amylase-based biocatalyst developed by researchers at the Central Leather Research Institute (CSIR-CLRI), Chennai, helps in processing leather in an environment-friendly way and also drastically cutting the time taken to process the skin at the pre-tanning stage. Pre-tanning process generates 60-70% of total pollution during processing.

Reduced effluents

The quantum of effluent discharge is considerably cut as there is threefold reduction in water usage when the biocatalyst is used. In particular, the amount of chromium that gets absorbed is more leading to less chromium discharge into the environment. Chromium is used for increasing the stability of the collagen through cross-linking. Since no chemicals are used, the chemical oxygen demand drops by about 35% while the total solid effluent load reduces by over 50%.

The reason why less chromium and water are required at the pre-tanning stage when the biocatalyst is used is primarily because of the 120-fold higher binding of the biocatalyst to the glycan sugar (glycosaminoglycan) present predominantly in the skin. Once the catalysts binds to the sugar, it selectively breaks down (hydrolysis) the sugar thus opening up the skin fibre. The results were published in the journal Green Chemistry.

Quick hydrolysis

“The binding and hydrolysis happens rapidly. It takes just 10 minutes to open the fibres when the biocatalyst that we engineered is used. Traditionally, enzymes take three-to-four hours to open the fibres. If lime and sulphate are used it takes 12 hours to complete the process,” says Niraikulam Ayyadurai from the Department of Biochemistry and Biotechnology at CLRI and corresponding author of the paper.

Not only is the process of opening the fibres quicker, the biocatalyst also penetrates deep into the skin unlike the traditionally used enzymes. Deep penetration of the biocatalyst has two advantages — it is sufficient to use less amount of chromium to increase the stability of the collagen and the quality of the finished leather also becomes superior. About 21% of the chromium used gets absorbed by the skin, which is far more than when other enzymes or chemical-based methods are used, leading to reduced chromium in the effluent discharge.

The biocatalyst is stable even at a high temperature of 90 degree C and pH 10 and so up to 95% of the enzyme can be recovered after a single process and reused.

Genetic engineering

The normal amylase enzyme has limited efficiency to bind to the substrate leading to reduced ability to open the skin fibre. So the team led by Dr. Ayyadurai resorted to genetic code. “The genetic code engineering allows us to introduce new chemistry in the amylase enzyme thus improving the enzymatic properties,” he says.

“The tyrosine amino acid was computationally modified with extra groups such as amino, hydroxyl, fluorine and chlorine. We found the extra hydroxyl group provided more activity towards the skin glycan,” says Suryalakshmi Pandurangan from CLRI and the first author of the paper. “By modifying the tyrosine amino acid we changed the property of amylase enzyme.”

The amylase gene was isolated from Bacillus licheniformis and made to express in E. coli. Large-scale production and even manipulation of the enzyme is possible when the enzyme is expressed by E. coli. It also becomes cheaper to produce the enzyme through the fermentation route.

Various medications have been tried for the disease, but with little success

The disease tuberculosis (TB) has been with us since around 10,000 years ago, when we humans began living in communities. We in India have known it since ancient times, just as the people in Western Asia have. Sanskrit texts dating back to 1500 BCE knew it and called it Sosha. Sushruta Samhita (ca. 600 BC) recommended that the disease be treated with breast milk, alcohol and rest. The compendium called Madhukosa (ca. 900 AD) described the disease as Yaksma or consumption .That the disease is transmitted from to person (even animals to humans) through sputum and cough was also known. Various medications were tried with little success. It was in 1882 that the German microbiologist H.H. Robert Koch discovered that the disease is caused by a germ he called Mycobacterium tuberculosis (or Mtb) for which he was awarded the Nobel Prize in 1905. He also attempted to find drugs to treat TB and came out with a product he called tuberculin, though with little success. Since then, drugs against TB have been successfully marketed, e.g., Rifampicin, Isoniazid, Pyrazinamide and the latest ones Pretomanid and Bedaquiline. The Indian government is using most or all of them to treat millions of TB cases every year.

Prevention is better than cure. The field immunology attempts to practice this proverb and tries to stop the invading germs from entering the body and causing disease. Success in producing such a product which can provide immunity against TB came about through the work of two French bacteriologists, Albert Calmette and Camile Guerin during 1908-1921. They came out with the product called Bacillus Calmette-Guerin or BCG. When BCG was injected in the human’s body as a vaccine, it provided immunity against the attack of Mtb. The first TB vaccine was thus born and is being used across the world even today on infants (and children below 15), affording them protection against TB. Remarkably, the protection lasts for as long as 20 years, as recent studies have shown. India has been using BCG vaccine with success for decades now.

Breathe in the vaccine

However, it has come to be known that while BCG is good for children, it may not be as effective in adults, who get TB of the lungs (pulmonary) more often, as opposed to in the intestines, bones or the urinary tract. It is also not effective if a person is affected by other diseases (for example, AIDS) and thus immune-compromised. Also, occasionally people react to BCG with some fever and also skin itching at the injection spot, making it uncomfortable to use. There is thus the need for alternative vaccines, which can work by themselves and also act as boosters for BCG. It will also help if these new vaccines are easier delivered in other modes, rather than by injection.

An exciting way to deliver vaccines was initiated almost 50 years ago and involved inhaling it through the nose into the lungs or the pulmonary route of vaccination. It was first tried in England in 1951 on a flock of chicken through aerosol vaccination against a virus. It was later tried with BCG in 1968, in guinea pigs and a few humans. In Mexico, they succeeded in immunising school-children against measles during 1988-1990, and later by others on TB (see review by Contreras, Awasthi, Hanif and Hickey: Inhaled vaccines for the prevention of TB 2012; Mycobacterial Disease S1:002. doi: 10.4172/2161-1068 S1-002). It is also worth noting that using this route needs no needles, does not need clinically trained persons, cuts down waste and is lower in cost.

When the pathogenic organism invades the body, it uses a molecule to pierce through and, using the material therein, to multiply and cause havoc. The host (scientists are polite!) body in turn fights back by using its immunity apparatus. The so-called B Cells there synthesise proteins called antibodies which bind with the invader and disable it. Plus, the host stores this mechanism for future, in case the pathogen attacks at a later time. This is the basis behind vaccination, which is good for years.

Actually, the whole germ is not needed for antibody generation. Even a part of the molecule (the “business region” or the epitope) suffices for the generation of antibodies by the B Cells. The immune apparatus has another class, called T cells which work in tandem with B cells. Molecules in T cells help “kill” the invaders. Here again, we do not need the whole molecule, but that part which helps in the process, as “adjuvants” for immune response.

A group at the University of Sydney in Australia has been creatively using these three principles to generate an inhalable vaccine against TB. Their publication appears in the latest issue of Journal of Medicinal Chemistry (Ashhurst. A, et al., J Med Chem. 2019 Aug 16. doi: 10.1021/acs.jmedchem.9b00832.). They chemically synthesised a part of the molecules of T cells as adjuvants on one hand and linked it chemically with the epitope part of Mtb (which too they synthesised in the lab). The so-synthesised product (call it compound I) was then administered through the nose of mice. The mice were then infected with Mtb and, after a few weeks the lungs and spleens of the infected mice tested. The bacterial load was found to be substantially low proving that aerosol administration of I was protective as a vaccine candidate. Also, the inhalation route of administering the vaccine candidate was seen to be better than the injection route. This should make children and nervous adults happy!

dbala@lvpei.org

The hydrogel reduces the cell size of the E. coli and disrupts its cell membrane

Tunable: By changing the boronic acid component used in the hydrogel, a large number of hydrogels with different bacteria-killing properties can be made

Researchers at the Indian Association for the Cultivation of Science (IACS) Kolkata have used a using naturally occurring nucleoside molecule cytidine to self-assemble into a hydrogel in the presence of silver acetate and phenyl boronic acid. The hydrogel possessing i-motif DNA-like structure was found to exhibit antibacterial activity against Gram-negative bacterial strains such as E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and multidrug-resistant Morganella morganii.

Non-toxic

While silver is known to have antibacterial property, it may not be used due to its toxicity. But when silver acetate was incorporated in the hydrogel the toxicity was reduced and thus suitable for treating bacterial infections. The hydrogel containing silver acetate was found to be non-toxic to normal kidney epithelial cells and red blood cells.

The research team led by Prof. Jyotirmayee Dash from the School of Chemical Sciences, IACS, found that the silver-containing hydrogel was capable of killing E. coli. The hydrogel reduced the cell size of the E. coli and disrupted its cell membrane, leading to leakage of cellular contents. The results of the study were published in the journal ACS Applied Bio Materials.

“The hydrogel can be tuned to change the anti-bacterial activity,” says Prof. Dash. “By changing the boronic acid component used in the hydrogel we can prepare a large number of hydrogels with different bacterial killing properties.”

Physical properties

Aside from self-assembling, the hydrogel exhibits thixotropic property — changes from a gel to a sol phase when subjected to mechanical shaking or stirring. When allowed to stand, it returns to its original gel phase. The hydrogel is also pH responsive. While being stable between pH 3 and 6, it becomes unstable at more acidic or alkaline pH.

“So the hydrogel can be used for drug delivery applications by using external stimuli such as mechanical stress or by changing the pH,” says Prof. Dash. The low pH of the gel is also another contributing factor for its intrinsic antibacterial activities.

The researchers loaded the hydrogel with cancer drug doxorubicin and made the gel release the drug by subjecting it to mechanical shearing and changing the pH. “We tested the drug-delivery property and in cellulo activities are currently under progress.

IIFPT, Thanjavur, scientists have 3D-printed a nutritious snack using millets, green gram, fried gram and ajwain seeds

Baked differently: The printed snack is microwave dried for best results.

Scientists have used 3D-printing to make automobile parts and prosthetics before but now 3D-printing food is becoming a reality. Researchers from the Indian Institute of Food Processing Technology (IIFPT), Thanjavur, have printed a nutritious snack using millets, green gram, fried gram and ajwain seeds. Taking just five to seven minutes to print, followed by a microwave drying process, this technology may help in customising food according to the individuals’ nutritional requirements.

Size of a mixie

“The printer is approximately the size of a mixie, weighing below 8 kg and can be carried around. It was also indigenously developed and completely fabricated in India. This brings down the cost to less than Rs.75,000, while most printers in the market are expensive and cannot be conveniently used for multi-material food printing applications,” says C. Anandharamakrishnan, Director of IIFPT and corresponding author of the paper published in the Food and Bioprocess Technology.

Efficient method

All the raw materials were hot-air dried, ground to fine particles and sieved to about 0.2 mm. Adding salt, spices, distilled water, they are made it into a paste and fed into the 3D-printer.

The flow, temperature, printer nozzle size and printing rate or speed was optimised after several trials. For further treatment of the printed snack, the researchers tried deep frying, hot air drying and microwave drying and found that the latter was the most efficient method.

High protein, fibre

The snack was also analysed for its colour, texture and taste. They also found that the snack had high protein and fibre content. “We can customise the nutrient content according to the need of the person. We tried this at a local school where we printed in shapes loved by children so that we can give them high nutrient food,” adds Dr. Anandharamakrishnan.

Earlier this year, the team had earlier made egg yolk and egg white into a printable form and studied its material characteristics, and optimised the printing conditions. This work was published in the Journal of Food Engineering.

Early stages

According to a scientist working in 3D-printing, food printing is in early stages in India, because to print food the dynamics and mechanics need to be fully understood. Though this may not be a solution to any food crisis or help altering the food manufacturing process, it may prepare ourselves for the future, he says. Perhaps one day this method may help print food at the International Space Station or any such environment. Instead of increasing the shelf life, printing food when and where needed can be a better option.

Yeast cells grew into groups with different metabolism, which were spatially organised

Variation: Fluorescent reporter genes were present in all cells but only some cells show the colour. The cells show spatial organisation.Sriram Varahan/Sunil LaxmanSriram Varahan

Bangalore-based researchers have shown that simple biochemical processes drive single-celled organisms to differentiate and become varied communities of cells having different metabolism. This study can help us understand how multicellularity develops.

The group studied the common baker’s yeast (Saccharomyces cerevisiae) to show how multicellularity can emerge in single-celled microbes due to merely biochemical effects. The experiment starts with yeast cells that are similar in all respects — they all produce a sugar (trehalose) from available raw materials. As the colonies mature and the concentration of trehalose increases, some of the cells start using up the sugar, thereby slowly bringing about a balance in the concentration of trehalose. In the process, this results in the development of cells with two different types of metabolism – cells that produce trehalose and those that use up trehalose. These new cells are confined geographically to some areas within the colony. Such characteristics are necessary for the development of multicellularity. The study has been published in the journal eLife.

When the colony was allowed to mature, the researchers found that the cells with different properties formed into groups across the colony.

Light and dark cells

“We just arbitrarily called these cells 'light' and 'dark' based on how they appeared under a light microscope,” says Sriram Varahan of Institute of Stem Cell Science and Regenerative Medicine (inStem), Bengaluru. “Subsequently, with more characterisation, we found that these cells have very different metabolism and properties,” he adds.

The researchers integrated three different approaches. Sunil Laxman of inStem explains: “We used many microscopic approaches to characterise the cells. We also used highly sensitive analytical chemistry approaches (mass spectrometry) to identify all the metabolic processes, and show that this new sugar is made, builds up, and then when used by some cells helps them to switch to a new state.”

Mass spectrometry

As the team had small quantities of experimental material, they used mass spectrometry to analyse and differentiate between the different types of cells.

“Our collaborators Dr. Sandeep Krishna and Vaibhhav Sinha from National Centre for Biological Sciences made a theoretical model that can predict this entire colony development, using these biochemical processes,” says Dr Laxman.

Many microbes can form biofilms, in which cells with different properties are together in a group. These biofilms play a role in many diseases caused by bacteria and fungi. This study explains how such biofilms can form, suggesting a means to control the growth of biofilms.

The study has thrown open several questions for the team. “We need to figure out how cells put out this sugar; how cells start to break down the trehalose; whether a minimum number of cells is needed for this to happen,” says Dr Laxman.

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