Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Aspergillus niger

The lignin peroxidase (isoenzyme H8) of Phanerochaete chrysosporium was expressed in Aspergillus niger under the control of plant nopaline synthase (NOS) promoter and terminator. H8 mRNA was produced in this heterologous system. Western blot analysis showed that the recombinant protein was of size similar to the native H8. The extracellular lignin peroxidase activity in these primary constructs was positive, though weak (1.12 nKat mg^–1).     

Preparation and testing of Sardinella peptones: application to lipase production by Staphylococcus sp

Production of lipase by Staphylococcus sp. in media containing fish peptones from sardinelle (Sardinella aurita) prepared in the laboratory was studied. Lipase production is strongly affected by lipids present in fish flours. Fish peptones prepared from dIgresed whole flesh was an excellent substrate for lipase production. A comparison of lipase production in media containing fish peptones or high quality commercial peptones indicated that fish peptones enhanced enzyme formation.     

Scorpion digestive lipase: a member of a new invertebrate's lipase group presenting novel characteristics

Unlike classical digestive lipases, the scorpion digestive lipase (SDL) has a strong basic character. The SDL activity's optimal pH, when using tributyrin or olive oil as substrate, was 9.0. Added to that, the estimated isoelectric point of the native SDL using the electrofocusing technique, was found to be higher than 9.6. To our knowledge, this is the first report of an animal digestive lipase having such a basic character. When olive oil was used as substrate, SDL was shown to be insensitive to the presence of amphiphilic proteins such as bovine serum albumin (BSA). Furthermore, the hydrolysis was found to be specifically dependent on the presence of Ca(2+) ions, since no significant SDL activity was detected in the presence of ions chelator such as EDTA. Nevertheless, the SDL does not require Ca(2+) to trigger the hydrolysis of tributyrin emulsion. Interestingly Zn(2+) and Cu(2+) ions act as strong inhibitors of SDL activity when using tributyrin as substrate. An internal chymotryptic cleavage of SDL generated two fragments of 28 and 25 kDa having the same N-terminal sequence. This sequence of 19 residues does not share any homology with known animal and microbial lipases. Polyclonal antibodies directed against SDL (pAbs anti-SDL) failed to recognise ostrich pancreatic and dog gastric lipases (OPL and rDGL). Moreover, both pAbs anti-OPL and anti-rDGL failed to immunoreact with SDL. These immunological as well as distinct biochemical properties strengthen the idea that SDL appears to belong to a new invertebrate's lipase group.     

Crab digestive lipase acting at high temperature: purification and biochemical characterization

In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.     

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.     

Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae

The effect of γ-ray irradiation on alcohol-dehydrogenase activity of yeast was investigated. The results suggested that low doses of γ-ray (10 and 20 Gy) significantly increased the enzyme activity. This work also describes the impact of irradiation on immobilization efficiency of biocatalyst entrapped on to alginate gel beads. When yeast irradiated to a dose of 20 Gy was immobilized, ADH stability was improved up to 1.4 times at 45 °C compared to the immobilized non-irradiated cells. Also, the irradiated biocatalyst, when immobilized, can be reused more than eight times in oxidation reaction of ethanol. This preparation also permitted to reach high yields of immobilization (79%) and activity (88%).     

Re‐programming of gene expression in the CS8 rice line over‐expressing ADPglucose pyrophosphorylase induces a suppressor of starch biosynthesis

The CS8 transgenic rice (Oryza sativa L.) lines expressing an up‐regulated glgC gene produced higher levels of ADPglucose (ADPglc), the substrate for starch synthases. However, the increase in grain weight was much less than the increase in ADPglc levels suggesting one or more downstream rate‐limiting steps. Endosperm starch levels were not further enhanced in double transgenic plants expressing both glgC and the maize brittle‐1 gene, the latter responsible for transport of ADPglc into the amyloplast. These studies demonstrate that critical processes within the amyloplast stroma restrict maximum carbon flow into starch. RNA‐seq analysis showed extensive re‐programming of gene expression in the CS8 with 2073 genes up‐regulated and 140 down‐regulated. One conspicuous gene, up‐regulated ~15‐fold, coded for a biochemically uncharacterized starch binding domain‐containing protein (SBDCP1) possessing a plastid transit peptide. Confocal microscopy and transmission electron microscopy analysis confirmed that SBDCP1 was located in the amyloplasts. Reciprocal immunoprecipitation and pull‐down assays indicated an interaction between SBDCP1 and starch synthase IIIa (SSIIIa), which was down‐regulated at the protein level in the CS8 line. Furthermore, binding by SBDCP1 inhibited SSIIIa starch polymerization activity in a non‐competitive manner. Surprisingly, artificial microRNA gene suppression of SBDCP1 restored protein expression levels of SSIIIa in the CS8 line resulting in starch with lower amylose content and increased amylopectin chains with a higher degree of polymerization. Collectively, our results support the involvement of additional non‐enzymatic factors such as SBDCP in starch biosynthesis.