Efficient Expression of Mono- and Diacylglycerol Lipase Gene from<i>Penicillium camembertii</i>U-150 in<i>Aspergillus oryzae</i>under the Control of Its Own Promoter

Yamaguchi, Shotaro; Takeuchi, Kikuko; Mase, Tamio; Matsuura, Akira

doi:10.1271/bbb.61.800

Cited by 11 publications

(7 citation statements)

References 8 publications

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“…Site-Directed Mutagenesis of Bilirubin Oxidase-The pNBC1 plasmid harbouring the M. verrucaria BO gene, diaminoacyl lipase (mdlA) promoter, and Aspergillus oryzae nitrate reductase gene (niaD) as a selection marker was constructed as reported earlier (14). In vitro mutagenesis was achieved by PCR using two primers, which have the recognition site of the restriction enzyme and sets of mutagenic oligonucleotides, as follows.…”

Section: Methodsmentioning

confidence: 99%

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

Shimizu¹

2003

Journal of Biochemistry

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Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.

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Section: Methodsmentioning

confidence: 99%

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

Shimizu¹

2003

Journal of Biochemistry

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“…In terms of overall structure, these molecules are very different from the fungal enzyme. Whereas the fungal enzyme is a single catalytic domain that is normally secreted into culture media [43], the newly identified enzymes have a four transmembrane (4TM) domain followed by a catalytic domain predicted to be intracellular (details given below). The 4TM lipases harbour a consensus serine lipase motif, and they were novel and unrelated to any characterized serine lipase, including MAGL, the enzyme responsible for hydrolysis of 2-AG to AA [44].…”

Section: Cloning Of Diacylglycerol Lipase A/bmentioning

confidence: 99%

The diacylglycerol lipases: structure, regulation and roles in and beyond endocannabinoid signalling

Reisenberg

Singh

Williams

et al. 2012

Phil. Trans. R. Soc. B

128

121

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The diacylglycerol lipases (DAGLs) hydrolyse diacylglycerol to generate 2-arachidonoylglycerol (2-AG), the most abundant ligand for the CB 1 and CB 2 cannabinoid receptors in the body. DAGL-dependent endocannabinoid signalling regulates axonal growth and guidance during development, and is required for the generation and migration of new neurons in the adult brain. At developed synapses, 2-AG released from postsynaptic terminals acts back on presynaptic CB 1 receptors to inhibit the secretion of both excitatory and inhibitory neurotransmitters, with this DAGL-dependent synaptic plasticity operating throughout the nervous system. Importantly, the DAGLs have functions that do not involve cannabinoid receptors. For example, 2-AG is the precursor of arachidonic acid in a pathway that maintains the level of this essential lipid in the brain and other organs. This pathway also drives the cyclooxygenase-dependent generation of inflammatory prostaglandins in the brain, which has recently been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. Remarkably, we still know very little about the mechanisms that regulate DAGL activity-however, key insights can be gleaned by homology modelling against other a/b hydrolases and from a detailed examination of published proteomic studies and other databases. These identify a regulatory loop with a highly conserved signature motif, as well as phosphorylation and palmitoylation as post-translational mechanisms likely to regulate function.

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“…Mg MDL2 exhibited high sequence identity with the SMG1 (67%). It shares only 20%, 20% and 19% sequence identity with those of DAG-like lipases from P. camembertii U-150 [ 5 ], P. cyclopium [ 6 ] and A. oryzae [ 9 ] ( Figure 1 ), respectively, while Mg MDL2 has a relative high sequence identity with that of TAG-lipases from R. miehei (28%) [ 14 ] and Rhizopus oryzae ( R. oryzae ) (24%) [ 15 ].…”

Section: Resultsmentioning

confidence: 99%

“…To date, few lipases including Penicillium camembertii ( P. camembertii ) U-150 [ 5 ], Penicillium cyclopium ( P. cyclopium ) [ 6 ], Fusarium sp. YM-30 [ 7 ], Aspergillus oryzae ( A. oryzae ) [ 8 , 9 ] and Malassezia globosa ( M. globosa ) [ 10 ], have been experimentally characterized as DAG-like lipases.…”

Section: Introductionmentioning

confidence: 99%

Biochemical Properties and Structure Analysis of a DAG-Like Lipase from Malassezia globosa

Lan

Yang

et al. 2015

IJMS

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Diacylglycerol (DAG)-like lipases are found to play an important role in the life sciences and industrial fields. A putative DAG-like lipase (MgMDL2) from Malassezia globosa was cloned and expressed in recombinant Pichia pastoris. The recombinant MgMDL2 was expressed as a glycosylated protein and purified into homogeneity by anion exchange chromatography. The activity of recombinant MgMDL2 was optimal at 15 °C and pH 6.0, and it keeps over 50% of relative activity at 5 °C, suggesting that MgMDL2 was a cold active lipase. MgMDL2 retained over 80% of initial activity after incubation at 30 and 40 °C for 2.5 h, but it was not stable at 50 °C. Incubation of methanol and ethanol at a concentration of 30% for 2 h did not affect the recombinant enzyme activity, while metal ions, including Ca2+, Mn2+ and Ni2+, sharply inhibited the MgMDL2 activity at 5 mM by 42%, 35% and 36%, respectively. MgMDL2 exhibited a preference for medium chain-length esters with highest activity toward p-nitrophenyl caprylate, while it was active on mono- and diacylglycerol but not on triacylglycerol, indicating that it was a typical DAG-like lipase. By homology modeling, Phe278 was predicted to be involved in the preference of MgMDL2 for monoacyl- and diacyl-glyceride substrates, but not triglycerides.

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Efficient Expression of Mono- and Diacylglycerol Lipase Gene fromPenicillium camembertiiU-150 inAspergillus oryzaeunder the Control of Its Own Promoter

Cited by 11 publications

References 8 publications

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

The diacylglycerol lipases: structure, regulation and roles in and beyond endocannabinoid signalling

Biochemical Properties and Structure Analysis of a DAG-Like Lipase from Malassezia globosa

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