Journal of Bioprocessing & Biotechniques

The β-L-arabinofuranosidase from Bifidobacterium longum JCM 1217 (HypBA1), a DUF1680 family member, was recently characterized and classified to the glycoside hydrolase family 127 (GH127) by CAZy. The HypBA1 exerts exo-glycosidase activity to hydrolyze β-1,2-linked arabinofuranose disaccharides from non-reducing end into individual L-arabinoses. In this study, the crystal structures of HypBA1 and its complex with L-arabinose and Zn²⁺ ion were determined at 2.23-2.78 Å resolution. HypBA1 consists of three domains, denoted N-, S- and C-domain. The N-domain (residues 1-5 and 434-538) and C-domain (residues 539-658) adopt β-jellyroll architectures, and the S-domain (residues 6-433) adopts an (α/α)6-barrel fold. HypBA1 utilizes the S- and C-domain to form a functional dimer. The complex structure suggests that the catalytic core lies in the S-domain where Cys417 and Glu322 serve as nucleophile and general acid/base, respectively, to cleave the glycosidic bonds via a retaining mechanism. The enzyme contains a restricted carbohydrate-binding cleft, which accommodates shorter arabino oligosaccharides exclusively. In addition to the complex crystal structures, we have one more interesting crystal which contains the apo HypBA1 structure without Zn²⁺ ion. In this structure, the Cys417-containing loop is shifted away due to the disappearance of all coordinate bonds in the absence of Zn²⁺ ion. Cys417 is thus diverted from the attack position, and probably is also protonated, disabling its role as the nucleophile. Therefore, Zn²⁺ ion is indeed involved in the catalytic reaction through maintaining the proper configuration of active site. Thus the unique catalytic mechanism of GH127 enzymes is now well elucidated.

The need for the contained handling and processing of pharmaceutical materials and pathogens is on the rise due to the increased focus on health and safety and the growing interest in the development of High Potency Active Pharmaceutical Ingredients (HPAPIs). A wide range of containment equipment is available on the market to meet the processing and operational needs of development and manufacturing. Selecting suitable equipment, however, requires an in-depth understanding of containment aspects in order to avoid operational hazards and large capital investments resulting from an ‘oversizing’ approach

All bio-containment guidelines and inspection documents set requirements for airtight containment boundaries around biohazards. Current international containment guidelines only scratch the surface for biosafety level 3 (BSL-3) and the available tests proposed are not entirely suitable for such facilities. Some steps in the right direction have been taken, notably as outlined in the standards put forth in the Australia-New Zealand model for their Physical Containment 3 designation.

C1-Esterase Inhibitor (C1-INH) is a protein present in human plasma and is a member of serine protease inhibitors super family. C1-INH is a molecule with lot of therapeutic importance and is widely used in the treatment of Angioedema and in the current research an improved purification scheme is developed to fractionate C1-INH from human plasma. By a series of chromatography steps C1-INH is purified to homogeneity. A simple three step chromatography procedure has been described excluding the more inefficient or cumbersome methods like precipitation or affinity capture. The C1-INH purified by this scheme is characterized along with the reference molecule. C1-INH produced by this method is a single-chain glycoprotein with a molecular weight of 85-93 KDa with increased purity and yields of 100-120 mg/L of plasma. The chromatography steps are easily scalable and the process is economical and adaptable for therapeutic protein manufacture.

Fructose is a potential additive that elevates a supply of electrons to hydrogenase for hydrogen production in cyanobacteria. A series of dark anaerobic hydrogen production experiments was performed to evaluate the validity of this assumption, in which fructose from 0 to 110 mmol/L was added to HEPES buffer solution on which a glucose tolerant mutant of unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (GT strain) was incubated. Despite the reported knowledge that fructose was an inhibitor for photoheterotrophic growth of Synechocystis cells, GT strain assimilated fructose and represented a limited heterotrophic growth on fructose in HEPES buffer solution under dark anaerobic condition. The initial hydrogen production rate that was 0.025 mmol/L h in run without fructose increased to 0.0917 mmol/L h in run with 60-83 mmol/L fructose. The associated increase in the initial amount of endogenous glucose from 0.22 mmol/L in run without fructose to 0.36 mmol/L in run with 50 mmol/L fructose was observed. Fructose released the complete suppression of hydrogen production by nitrate. This work presents the first experimental evidence that cells of GT strain are able to assimilate fructose for cell growth in dark anaerobic condition. Our results show that hydrogen production in Synechocystis sp. strain can be significantly elevated by a proper addition of fructose to dark anaerobic HEPES buffer solution.

AuTiO2 nanomaterial was used for solar photocatalytic disinfection of gram negative Escherichia coli and gram positive Staphylococci aureus. In this study, time of disinfection, light intensity, concentration of nanomaterial and bacterial concentration effects has been investigated. Prepared nanomaterial characterized using Scanning Electron Microscopy (SEM), X-Ray Diffractometry (XRD). AuTiO2 samples have shown antibacterial effect when irradiated in solar light. The bactericidal effect may be due to Au ions and photocatalysis of TiO2. The photocatalytic inactivation rate constant, k for each dose were obtained from their respective inactivation curve over a 2 hours incubation period.

The current work is enlightened about a cost effective bioprocess using one factor at a time approach for the production of bio-surfactant through solid state fermentation. A fungal strain Penicillium chrysogenum SNP5 isolated from grease contaminated soil was reconnoitered for the production of bio-surfactant. Various physiochemical parameters i.e., substrate composition, nitrogen supplements, extraction media and pH were optimized in order to optimized the production in terms of emulsification index and oil displacement assay. Maximum oil displacement area produced using grease waste and wheat bran (1:1 w/w), waste cooking oil and wheat bran (1:1 v/w) as a substrate were 3.5 cm and 5 cm, respectively. Whereas, considered values for emulsification activity with oil and diesel were 43% and 22% during optimization of substrate composition. Variable ratios of grease waste and wheat bran were capable to enhance the emulsification activity with oil and diesel up to 45% and 24% in presence of grease and wheat bran (1.5:1). The strain also showed enhancement of emulsification activity 45% and 23% with oil and diesel respectively to utilized yeast extract as a nitrogen source and the highest emulsification activity 38% in diesel, 47% in oil and oil displacement 5.5 cm was found at pH 8 with grease and wheat bran as a substrate. Preliminary characterizations by thin layer chromatography showed that the bio-surfactant was lipopeptide in nature and was also confirmed through FTIR analysis. Metabolization of industrial grease waste through solid state fermentation has never been reported before for the production of biosurfactants therefore would be applicable in petroleum and biodiesel industry. The partially purified biosurfactants was further investigated for antimicrobial activity and enhanced oil recovery. It displayed effective zones of inhibition against both gram +ve (1.67 cm) and gram –ve (1.93 cm) as well as 16.5% enhanced recovery of oil. Both results also give a positive support to its role in pharmaceuticals as well as in petroleum and oil industry.

The obromine is a metabolic intermediate produced in caffeine degradation pathway by many bacterial species, which has potential applications in food and pharmaceutical industries. Conventional methods of Theobromine production from xanthine involve harsh physical and chemical conditions which are harmful to the environment. To overcome this, we employed biotechnological route to convert caffeine to theobromine by single demethylation reaction using induced cells of Pseudomonas sp. Initially we screened various divalent metal ions for the production of Theobromine by Pseudomonas sp. from caffeine. Co2+ and Ni2+ accumulates 400 and 100 mg/l of theobromine under initial reaction conditions (2 g/l caffeine, 8 g/l cell loading, pH 7.0,30°C). Co2+ was chosen for further optimization of reaction conditions for Theobromine production using response surface methodology. Data were fitted into a quadratic model and the optimal condition for theobromine production was found to be 3.2 g/l caffeine, 11.3 g/l initial cell loading and pH 7.0. Quadratic regression models were validated at the optimized conditions and the experimental theobromine produced 689.7 mg/l corresponds to the model predicted theobromine 729.4 mg/l. Theobromine production was further improved to 1.08 ± 0.10 g/l by optimizing the reaction temperature. This study reports highest production of theobromine from caffeine using induced cells of Pseudomonas sp. Induced cells are better suited for metabolite production as it is metabolically very active and can be re-used several times. Optimization of reactor parameters will enable us to make microbial production of theobromine feasible in industries at reduced cost.