బయోప్రాసెసింగ్ & బయోటెక్నిక్స్ జర్నల్

Poly (N-isopropylacrylamide) (PNIPAM) has been widely referenced as an alternative solution for proteolyticenzyme- based cell harvesting. However, the reversibility (i.e., consecutive attachment-detachment cycles) of PNIPAM for cell harvesting has not been studied. In this work, we synthesized PNIPAM-grafted glass surfaces to study the efficiency of fibroblastic cell detachment after iterative use. For robust quantification of thermally triggered cell detachment, an innovative wide-field lens free video microscope was introduced. The cell detachment efficiency was shown to decrease significantly after each cell culture experiment due to the loss of polymer brush thermosensitivity. The influence of the temperature variation rate on cell detachment from the re-used PNIPAM-grafted surfaces was further characterized. The cell detachment behavior on these substrates was shown to be quite similar to that on bare glass substrates. Furthermore, it was demonstrated that non-negligible cell detachment occurs and is modulated according to the temperature variation rate. This study aims to inform scientists using PNIPAM-grafted substrates for cell detachment about possible issues, to help scientists to more carefully monitor culture experiments, and to aid scientists in the interpretation of results obtained from cell detachment experiments performed on PNIPAM-grafted surfaces.

Background: To improve the diagnostic and clinical treatment of sleep disorders, the first important step is to identify or detect the sleep stages. Utilizing the conventional method-known as visual sleep stage scoring-is tedious and time-consuming. Therefore, there is a significant need to create or develop a new automatic sleep stage detection system to assist the sleep physician in evaluating the sleep stages of patients or non-patient subjects. The first aim of this study is to develop an algorithm for automatic sleep stage detection based on Electrooculography (EOG) signals. The second aim is to utilize sleep quality parameters to classify and screen Periodic Limb Movements of Sleep (PLMS) patients and Sleep Apnea Hypopnea Syndrome (SAHS) patients, as distinct from healthy control subjects. Methods: 10 patients with Periodic Limb Movements of Sleep (PLMS), 10 patients with Sleep Apnoea Hypopnea Syndrome (SAHS), and 10 healthy control subjects were utilised in this study. Several features were extracted from EOG signals such as cross-correlation, energy entropy, Shannon entropy and maximal amplitude value. K-Nearest Neighbour was used for the classification of sleep stages. Several polysomnographical (PSG) features were measured for screening and classification of the sleep disorders, such as the percentage of the sleep stages over the total time of sleep, the duration of the sleep stages, Sleep Latency (SL), and sleep efficacy. A decision tree analysis was utilised for identifying the three groups of subjects. Results: The overall accuracy, sensitivity and specificity of automatic sleep stage detection were 80.5%, 81.3% and 88.8%, respectively. The Cohen’s Kappa was 0.73. The performance of the classified sleep disorders showed an overall accuracy of 90%. The sensitivity and specificity were 90% and 95%. The Cohen’s Kappa was 0.85. Conclusion: One advantage of the automatic sleep stage detection method based on Electrooculography (EOG) signals is that it can be utilized with portable sleep stage recording instead of using a multichannel signal. Classification of sleep disorders based on the automatic system is an improvement, in that it can make the screening or diagnostic processes much faster and easier than with other methods.

A new endo-1,4-beta-xylanase gene (FJ380951), KRICT PX1, isolated from Paenibacillus sp. HPL-001, was expressed in E. coli. Enzyme purification, mutation, immobilization, and molecular simulation were conducted. The P1H1 mutant of one amino acid substitution (168Leu￫Gln) showing the strongest xylanase activity was selected among 250 mutant clones. The specific activity of the P1H1 xylanase was 53.3 U/mg protein at 50°C, an approximately fivefold increase compared to that of the original KRICT PX1 xylanase (10.25 U/mg protein) at the same condition of pH 5~10, and enhanced activity from 20.1 to 36.6 U/mg proteins at 60°C. The structural dynamics of the mutant P1H1 became stronger than that of the wild type, which could be eliminated by a helix deformation with the H-bond missing between Leu₁₆₈ and Asp₁₆₄ after mutation. Xylanase could be reused for 5 batches without significant loss of activity after being immobilized on surface functionalized silica-based mesoporous cellular foam. However, xylanase activity declined to 60% at the 10th batch. Further research on practical applications will be necessary for industrial usage.

Carbon Nanotubes (CNTs) had been synthesized by Double Stage Chemical Vapor Deposition (DS-CVD) at the gas phase with Hydrogen gas (H2) and acetylene (C2H2) as precursor gases. Moreover, the structure of CNTs, morphology and purity were characterized by using the Transmission Electron Microscope (TEM), Field Emission Scanning Electron Microscope (FESEM) and Thermogravimetric Analysis (TGA). Furthermore, the best conditions for the optimal CNT was reaction at temperature of 750°C, reaction time of 30 minutes, with gas flow rates H2 and C2H2 for 60 and 170 ml/min for respectively. The hydrochloric acid was used to purify produced CNT and then using the nitric acid and sulfuric treatment to functionalize. Moreover, functionalized CNTs, non-functionalized were used as column chromatographic media for skim latex protein purification. Thus, the results reveal that at pH7 and ionic strength 50 mM gives higher efficiency of protein removal from the skim latex (Graphical abstract (Figure 1)).

Graphical abstract: Synthesis and characterization of MWCNTs for protein purification.

The 35 bacterial strains were isolated from water and soil sample from Karnataka (India) and screened for their ability to secrete extracellular Alkaline Phosphatase (ALP). Among them, a potent strain Proteus mirabilis resembling to Proteus mirabilis strain M54 was confirmed by 16S rRNA gene sequencing, was selected for the optimization of enzyme production. The optimum production was at pH 7-8 and at temperature 35°C for 28 hrs incubation. The pNPP was the best substrate for the ALP production. This production was stimulated by using starch and casein as sources of carbon and nitrogen respectively. After purification by Ammonium sulphate precipitation, gel filtration and ion exchange chromatography, the fold purification was increase up to 9.7. The molar mass was estimated by 12% SDS PAGE and was found to be approximately 56 kDa and also confirmed by gel filtration studies (data not shown). The study holds significance as there are only few reports available on the optimisation protocols.

World is threaten to energy crisis which has advances research in bioenergy and specifically development of biofuels to replace petroleum products have increased the use of microbial enzyme like cellulases and xylanases as well as amylases for generation of reducing sugars for their conversion into bioethanol. Extensive research has been carried out in this view but alkaline cellulase production and molecular characterization is not studied in detail so far, this study will aid to achieve it. Optimization of fermentation parameters for production of cellulase was evaluated with the help of Response Surface Methodology (RSM) a statistical design, initial pH (9), moisture ratio (1:1) and incubation time (72 h) (run no.4) were found to be ideal parameters for optimum production of cellulase, substrate Jatropha seed cake without any pre-treatment was found to be an ideal source for cellulase production by Bacillus licheniformis under solid state fermentation. Cellulase gene of size 786 bp was isolated later using PCR techniques, confirmed with sequence analysis and ligated to pRSET A vector for the transformation to E. coli DH5α. Positive clones were identified and sequenced to justify the cloning. Sequence of Bacillus licheniformis endo-β-1,4-glucanase (Cel12A) gene showed 100% similarity with endoglucanase gene sequence from Bacillus licheniformis ATCC 14580 genome, shows successful cloning of Cel12A gene into pRSET A vector.

In this work, araujiain (enzymatic preparation obtained from the latex of Araujia hortorum fruits) was successfully immobilized on glyoxyl-agarose via multipoint covalent attachment. Thus, good efficiency of immobilization and high operational stability of immobilized enzyme were obtained. The activity of araujiain at alkaline pH was significantly improved after immobilization. In addition, immobilized araujiain also showed high activity and good stability, without significant loss in its activity, at temperatures between 37 and 60°C and in the presence of immiscible organic solvents. Immobilized araujiain also showed good performance in a mixture of 50% ethyl acetate in buffer, used for peptide synthesis, with better results than when the free enzyme was used as catalyst. These results indicate that immobilized araujiain via multipoint covalent attachment can be highly stabilized and this method might be used for practical applications of araujiain in hydrolytic and synthetic processes.

Hericium erinaceus is a medicinal mushroom producing biologically active metabolite erinacin A. In this research we compared fungal growth and metabolite production in different reactor types and different substrates with added casein peptone and NaCl. Cultivation of fungal biomass was performed in glass jars and horizontal stirred tank reactor. The cultivation in reactor was carried out at 24°C and airflow 5 L/min. Periodical mixing of N = 80 rpm, was used. Solid state substrate based on mixture of husked millet and paddy millet was used. For simultaneous determination of erinacine A and ergosterol contents in a single sample new analytical method was developed. The amount of biomass obtained in glass jars averaged at 100 mg/g ± 12 mg/g, while in horizontal mixing bioreactor it easily raised up to 350 mg/g. The highest erinacin A concentration was detected in substrate with 0.56% NaCl and 3.4% casein peptone content.