Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol.

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads® EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.

Methyl farnesoate synthesis in the lobster mandibular organ: the roles of HMG-CoA reductase and farnesoic acid O-methyltransferase.

Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ-inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT-mRNA levels after 5h. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar.

Characterization of a lipid activated CTP:phosphocholine cytidylyltransferase from Drosophila melanogaster.

CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6 x -His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V(max) value of 1352+/-159 nmol CDP-choline/min/mg, a K(m) value of 0.50+/-0.09 mM for phosphocholine, and a K' (Hill constant) value of 0.72+/-0.10 mM for CTP. The 6 x -His-tagged enzyme has similar properties with a V(max) value of 2254+/-253 nmol CDP-choline/min/mg, a K(m) value of 0.63+/-0.13 mM for phosphocholine and a K' for CTP equal to 0.81+/-0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators.

The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase.

In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.

3-hydroxy-3-methylglutaryl-coenzyme A reductase in the lobster mandibular organ: regulation by the eyestalk.

The mandibular organ (MO) of the lobster, Homarus americanus, produces the isoprenoid methyl farnesoate (MF), a compound related to insect juvenile hormone (JH). To better understand the synthesis and regulation of MF, we studied 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), the rate-limiting enzyme in isoprenoid biosynthesis. Lobster HMGR had a Km of 11.4 microM for HMG-CoA, a Km of 14.8 microM for NADPH, and was at least 2000-fold more selective for this cofactor than for NADH. Lovastatin and mevalonic acid inhibited HMGR, with KI values of 1.3 nM and 25.3 microM, respectively, whereas MF, farnesoic acid, cholesterol, 20-hydroxyecdysone, and progesterone had no effect. Approximately 75% of the HMGR activity in lobster MO was soluble. Similar levels of HMGR activity were observed in all regions of the MO. Eyestalk removal increased MF synthesis and the activity of farnesoic acid O-methyltransferase (FAOMeT, the final step in MF synthesis) in the MO by 10.7- and 5.7-fold, respectively, and caused a 3.1-fold increase of HMGR activity. Injection of the eyestalk ablated lobsters with an extract of two sinus glands (SG), a neuroendocrine organ in the eyestalk, decreased MF synthesis, FAOMeT activity and HMGR activity to 3, 8, and 20%, respectively, of the levels observed in saline-treated animals. The regulation of crustacean HMGR by the SG suggests that the lobster MO is a useful model system for investigating the cellular regulation of HMGR activity.