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Enzymatic cycling system (coupled dehydrogenase-catalyzed biosystem being composed of two elementary enzymatic reactions mediated by NAD(P)H + NAD(P)+) is industrially attractive for reducing prochiral carbonyl compounds to the corresponding chiral hydroxyl compounds. The reaction rate equation of the batch-wise biosystem was generally derived by ordered Bi Bi mechanism of two-substrate enzyme reaction on several reasonable assumptions. The rate equations of the batch-wise biosystem was generalized by transforming them into the dimensionless forms. The dimensionless forms were solved numerically. It was revealed that the batch-wise biosystem was generally made up of unique 3 phases, i.e., phases I, II and III. Phase I was very short transient so that the biosystem entered rapidly phase II. In phase II the consumption rate dynamically balanced with its formation rate so that the concentration of NAD(P)H was invariable with time (and hence NAD(P)+ concentration was, too). Phase III was substrate-exhausting phase, and the coenzyme concentration became finally only [NAD(P)+] or only [NAD(P)H] depending on the initial molar ratio of the prochiral carbonyl compound to the substrate of the coenzyme regeneration reaction ( [ S ] 0 / [ S ′ ] 0 ) > or [ S ] 0 / [ S ′ ] 0 1.0 , were proposed. As the main assumption irreversibility of the two elemental enzymatic reactions was discussed. Validity of the proposed rate equations was mentioned.

There is considerable interest in the development of simple methods for quantifying production of the biodegradable polyester poly(3-hydroxybutyrate) [P(3HB)] by bacteria. Cells of Cupriavidus necator were grown on agar medium containing different concentrations of glucose (10–25 g/L) as a sole carbon source. Trace amounts (100±5 μg) of dried C. necator cells were directly subjected to thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of tetramethylammonium hydroxide (TMAH). On the resulting chromatograms, a series of characteristic peaks, attributed to the THM products from poly(3-hydroxybutylate) accumulated in the bacterial cells, were clearly observed without any interfering component. Based on the peak intensities, the contents of P(3HB) in C. necator were determined precisely and rapidly without using any cumbersome sample pretreatment. Furthermore, the values of the P(3HB) contents coincided overall with those obtained by the conventional method involving solvent extraction followed by gravimetric determination.

Kinetics of batch-wise enzymatic cycling system (oxidoreductase-catalyzed reaction system involving enzyme-coupled cofactor regeneration) has been studied covering a broad range of the conserved total cofactor concentration, [C]0 (=NAD(P)+ + NAD(P)H), based on reasonable several assumptions. It is composed of two elementary reactions, i.e. product synthesis reaction and cofactor regeneration reaction, both of which have been expressed by Michaelis–Menten type rate equations. A novel dimensionless variable, r, has been introduced, which is defined as the concentration of one of the two cofactor components, [X] (NADH+ or NADPH+), divided by [C]0, i.e. r .e[X]/[C]0. The following results have been obtained. (1) The fundamental equation of the batch-wise enzymatic cycling system has been transformed to a differential equation whose formula is: dr/dT = N(r)/D(r) (N(r) and D(r) are quadratic equations of r having different coefficients). (2) It has been elucidated that the batch-wise enzymatic cycling s...

In order to produce microbiologically large amount of l (+)-lactic acid (LA) from glucose, batch and fed-batch (intermittent addition of sterilized glucose powder aseptically) cultures of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) whose mycelia were immobilized in situ within sponge-like cubic particles (3.5 mm edge long) were carried out at 37°C in a three baffled shake flask. Appropriately calculated amount of fine powdery calcium carbonate (CaCO 3 ) was added initially or intermittently to control pH of the culture liquids. High accumulations of LA (145 g/L and 231 g/L, in reality 176 g/L and 280 g/L as anhydrous calcium lactate) were achieved by a batch (glucose concentration = 150 g/L) and a fed-batch cultures (the initial glucose concentration = 150 g/L and the intermittent addition of glucose equivalent to 100 g/L). In these cultures the yields and productivities of LA were, 95.0%, 1.42 g/L·h and 92.5%, 1.83 g/L·h, respectively. Existence of considerable amounts of calcium lactate (Ca(LA) 2 ) as crystals in the culture broth was experimentally proved by two evidences: (i) heating up (70°C) followed by quick low centrifugal force to remove remaining CaCO 3 solids from culture broth and then cooling down (37°C) followed by incubation of the culture supernatant at 37°C to observe recrystallization of Ca(LA) 2 , and (ii) the measurement of solubility of Ca(LA) 2 in the culture media. It was conceptually discussed to be able to avoid the product inhibition by means of crystallization fermentation for the high accumulation of LA by R. oryzae .

An extracellular lipase was isolated from a culture filtrate of Fusarium oxysporum f. sp. lini SUF 402 by hydrophobic chromatography. Purity of the preparation was 38-fold and recovery yield was 32%. The molecular mass of the isolated enzyme was 30 kDa. The lipase had a sugar chain, and the N-terminal amino acid was modified. The optimun pH at 37°C was 7.0. The enzyme had higher activity toward p-nitrophenyl esters with long and middle chain fatty acids (C8-C18) than with a short chain fatty acid (C4). The enzyme was not inhibited by phenylmethylsulfonyl fluoride or 2-mercaptoethanol. In the case of the lipase, both the hydrolysis rate of tristearin and final concentration of fatty acid content were higher than those of triolein. The lipase was examined with respect to its ability of concentrating the n-3 polyunsaturated fatty acid (n-3 PUFA) content of partially hydrolyzed glycerides (TG and DG) obtained from two kinds of fish oil (cod liver oil and refined sardine oil). The lipase gave increases in n-3 PUFA contents as the hydrolysis progressed. The lipase concentrated only docosahexaenoic acid with little increase in icosapentaenoic acid. Maximal total n-3 PUFA contents were about 30% in both fish oils.

Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.

To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of bioinformatic analysis, high-throughput screening can expand its potential for exploring vast combinatorial sequence spaces of proteins.

A novel HPLC-based method for direct separation of the three isomers of mono-acid diacylglycerols (DAGs), i.e., 1,2-DAG, 2,3-DAG and 1,3-DAG, has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and a chiral stationary phase column) are connected in series. Two isomeric mixtures of DAGs (i.e., dicapryloylglycerol and dioleoylglycerol) and lipase-catalyzed reaction mixtures were successfully resolved on the tandem column HPLC system without any derivatization prior to the analysis. According to the established analytical method, stereoselectivity of two lipases toward mono-acid triacylglycerols in ethanolysis reaction was investigated. The tested enzymes were immobilized Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML). Analyses of the enantiomeric purity of 1,2-DAG and 2,3-DAG, generated as intermediates during the reaction, revealed that CALB and RML have sn-3 and sn-1 stereopreference, respectively.

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.

Microbial poly(3-hydroxyalkanoates) (PHAs) with fluorinated phenoxy side groups were produced by Pseudomonas putida when fluorophenoxyalkanoic acids were used as carbon sources. 11-(2-Fluorophenoxy)undecanoic acid (2FPUDA), 11-(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11-(2,4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11-(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA) were used as carbon sources in the present study. When cells were grown with 2,4DFPUDA, the production of homo poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was confirmed by NMR and GC/MS analyses. Fluorine atoms inserted into the side chain of the PHA dramatically affected its physical properties. In marked contrast to medium chain length (MCL) PHA, this fluorinated PHA was opaque, cream colored, and possessed greater crystallinity and a higher melting point (∼100 °C) than did the other MCL PHAs. Surface contact angle evaluation revealed that the PHA with two fluorine atoms possessed water-shedding properties. The number of substituted fluorine atoms in the carbon source affected cell growth and difluorine-substituted phenoxyalkanoic acids reduced cell growth, and polymer production compared to non-substituted phenoxyalkanoic acids. No polymeric materials were obtained using either 2,4,6TFPUDA or 2,3,4,5,6PFPUDA.

The Dam1 complex, also known as DASH complex, is the outer kinetochore protein complex of yeast that plays a crucial role in attachment of kinetochore to microtubule. The Dam1 complex is formed by at least nine proteins including Dam1p, Duo1p, Dad1p, Spc19p and Spc34p. In this study, domains of Spc34p that physically interact with other subunits of the complex were mapped using a high-throughput methodology. The method is a combination of two-hybrid screening of a random truncation library of the Spc34 gene and a unique PCR-based amplification that converge the selected DNA fragments to a few short fragments. Duo1p, Dam1p, Dad1p and Spc19p binding domains of Spc34p were mapped on M1-E59, M1-D47, M1-D47 or T207-E295 and S154-Q294, respectively. Most of the boundaries were located at less conserved regions among fungal Spc34p homologs, which is consistent with the boundaries of the putative secondary structures. The accuracy of the mapped domain boundaries was verified using truncated Spc34p polypeptides. The results and methodology we demonstrated herein not only shed light on the molecular architecture of the protein complex but also pave the road to the high-throughput identification of specific interaction domains of proteins whose possible interaction partners have been identified in genome-scale analyses.

A novel entirely in vitro strategy for generation and screening of a combinatorial protein library in an array format has been developed and is experimentally demonstrated. The strategy exploits virtues of PCR and in vitro coupled transcription/translation. Our new approach provides high-throughput construction and screening of the in vitro protein library, and compatibility with various selection methods.

We found that the affinity tag fused to the carboxyl (C-) terminal of a single-chain Fv (scFv) antibody was proteolytically degraded in a wheat germ cell-free protein synthesis system. The addition of two extra residues of glycine to the tail of the cMyc tag significantly increased the stability of the tag, suggesting that wheat endogenous carboxypeptidase(s) play a primary role in the C-terminal tag-specific degradation. In addition to the modification of the tag sequence, addition of diisopropyl fluorophosphate, which is known as an inhibitor of carboxypeptidases, prevented the cMyc tag sequence degradation. The effects of other protease inhibitors on the translation reaction and stability of the synthesized protein are also reported.

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) (GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.

By thein vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis andin vitro coupled transcription/translation withEscherichia coli S30 extract, S65 and E222 of green fluorescent protein ofAequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. Thein vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans , codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR , suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[( R )-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator. To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis. In this paper, Phe119 and Leu167, which are located in the acyl portion of the substrate-binding pocket of the lipase of B.cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis. The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation. The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate, p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates. Two mutant lipases, L167V and F119A/L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro. It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester.

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C22 residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene ( phaZ ) was identified near the PHA synthase gene ( phaC ) of P. denitrificans . Cell extract of Escherichia coli carrying lacZ–phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D (−)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg 2+ ion inhibited PHB degradation. However, the inhibitory effect by Mg 2+ ion was not observed using the cell extract of P. denitrificans .

A highly efficient enzymatic method for the synthesis of regioisomerically pure 1,3-dicapryloyl-2-docosahexaenoyl glycerol (CDC) in two steps was established. 2-Monoglyceride (2-MG) formation by ethanolysis of tridocosahexaenoylglycerol (DDD) with immobilized Candida antarctica lipase (Novozym 435) as catalyst was the key step of the synthesis. CDC was finally obtained by reesterification of 2-MG with ethylcaprylate (EtC) catalyzed by Rhizomucor miehei lipase (Lipozyme IM). The regiospecificity of Novozym 435 depended on the type of reaction and the initial composition of the reaction medium. It displayed strict 1,3-regiospecificity for ethanolysis at a high excess of ethanol in the reaction mixture although it displayed no regiospecificity in transesterification and esterification reactions. The highest yield of CDC (85.4%) was obtained by ethanolysis at a 4∶1 weight ratio of ethanol/DDD for 6 h followed by reesterification at a 20∶1 molar ratio of EtC/initial DDD for 1.5 h. The regioisomeric purity of CDC was 100%. Good results were obtained also for the synthesis of 1,3-dicapryloyl-eicosapentaenoylglycerol (CEC) by the same method: 84.2% yield and 99.8% regioisomeric purity at the same reactant ratios as above. The yield of the reesterification step and the regioisomeric purity of the product were influenced by the molar ratio of the reactants for both CDC and CEC syntheses: higher excess of EtC favored higher yields and regioisomeric purity of the products.

A novel multivariable control strategy is developed for alcohol (ethanol and n-pentanol) concentrations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(HB-co-HV), a biodegradable polymer by Paracoccus denitrificans ATCC 1774. This controller, which is developed to control the mole fraction of P(HB-co-HV), consists of two parts: one is for ethanol concentration control and the other is for mole fraction control, based on the concept of metabolic flux distribution control. A simple metabolic reaction (MR) model is constructed for flux distribution analysis. The relationship between mole ratio of specific consumption rate of the two alcohols (ethanol and n-pentanol) and the mole fraction of 3HV units in the polymer is linear. This result suggests that the split ratio at a branched point of 3-ketovaleryl-CoA in the P(HB-co-HV) synthetic pathway is constant for several fermentation conditions. When the mole fraction of 3HV units has a target value, the feed rate of n-pentanol becomes a function of the feed rate of ethanol and the set value of 3HV, based on the MR model. The mole fraction of 3HV units successfully reached the target value using this strategy. The mole fraction control strategy is combined with an optimal production strategy based on the optimal profile of the specific growth rate. The combined strategy is realized using multivariable controllers and P(3HB-co-3HV) production is maximized with a given value of mole fraction of 3HV units at the final step of fermentation.

A simple method for direct optical resolution of some asymmetric triacylglycerols (TGs) has been established. The method employs chiral-phase high-performance liquid chromatography (HPLC). An enantiomeric pair of TGs comprising 1-eicosapentaenoyl-2,3-dicapryroyl-sn-glycerol (ECC) and 1,2-dicapryroyl-3-eicosapentaenoyl-sn-glycerol (CCE) was resolved on a CHIRALCEL OF™ or on a CHIRALCEL OD™ column. The separation of another pair of asymmetric TGs, 1-docosahexaenoyl-2,3-dicapryroyl-sn-glycerol (DCC) and 1,2-dicapryroyl-3-docosahexaenoyl-sn-glycerol (CCD), was achieved with the CHIRALCEL OD column. The chiral-phase HPLC method in combination with silver-ion HPLC and high-temperature gas chromatography was used for monitoring two interesterification reactions, whose products were chiral TGs. Interesterification of tricapryloylglycerol with ethyleicosapentaenoate or with ethyldocosahexaenoate was performed using Rhizomucor miehei lipase as the catalyst. The products targeted were the asymmetric pair of TGs, ECC and CCE or DCC and CCD. The amounts of sn-1-substituted products (ECC or DCC) were greater than their sn-3-substituted counterparts (CCE or CCD) throughout the reaction period, suggesting that R. miehei lipase had a stereopreference towards the sn-1 position over the sn-3 position.

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.

To reduce the freezing point of sesame oil, the lipase-catalyzed interesterification of sesame oil in a solvent free system was studied. The lipase was immobilized on Celite and refined sesame oil was used as the only substrate for the reaction. After interesterification, the oil did not solidify at 0 degrees C even after 24 h, and even longer storage at 2-4 degrees C did not result in solidification. The change of physical behavior was investigated with a differential scanning calorimeter and X-ray diffraction, and reduction in the thermodynamic and crystallographic stability of the interesterified oil was demonstrated. The change in triacylglycerol species composition after the reaction was analyzed, showing that content of trisaturated acylglycerol was decreased.

Poly(3-hydroxyalkanoate) (PHA) with a thiophenoxy group at the end of the side chain was obtained from Pseudomonas putida 27N01, using 11-thiophenoxy undecanoic acid as the sole carbon source. NMR and GC/MS analyses indicated that the polymer consisted of 3-hydroxy-5-thiophenoxypentanoate as the primary monomer unit and 3-hydroxy-7-thiophenoxyheptanoate as a minor unit. The polymer had a white cream color and was elastometric. X-ray analysis suggested that the PHA had an amorphous-like diffraction pattern. The final dry cell concentration was 693 mg/L, and the PHA content of the dry cells was 19.5%. GPC measurement with polystyrene as a standard showed that the number-average molecular weight was 81 000 with a polydispersity of 1.8. The glass transition temperature was approximately 4 °C according to DSC. Thus, we were able to successfully characterize this PHA and to show that a sulfur atom can be incorporated into the bacterial polymer produced from P. putida 27N01.

Production of structured triacylglycerols (sTAG) containing eicosapentaenoic acid (EPA) at the sn-1 (or 3) position using Lipozyme in a solvent-free system was studied. Optimal water activity (a(w)) for the synthesis of the sTAG was investigated. Vacuum was applied to shift reaction equilibrium toward the synthesis reaction by removing by-products. During vacuum application, the water level of the reaction system was controlled at the optimal level by addition of a suitable amount of water at a predetermined interval. Intermittent periodic addition of a suitable amount of water into the reaction mixture made the reaction rate faster than that without adding water. A molar yield of 89.7% of the targeted sTAG was obtained after 16 h reaction with a(w) control during the vacuum application as compared with the yield of 87.0% after 24 h of reaction without a(w) control during the vacuum application.

A novel bioreactor using a hollow-fiber membrane was developed for the coupled transcription/ translation system using T7 RNA polymerase and Escherichia coli S30 extract. The large surface area per the reaction volume of the reactor assured rapid mass transfers of substrates into the reaction mixture and of wastes out from it across the membrane by their molecular diffusion. The flux was large enough to maintain nucleotide concentrations for more than 3 h, which increased the protein synthesis greatly. In addition, the T7 terminator sequence downstream from the reporter genes was found to increase the synthesized protein significantly, especially when the product of polymerase chain reaction (PCR) was used as a template. Implementation of this finding and use of the bioreactor developed multiplied the productivity of protein by the in vitro direct expression from PCR template.

The lipase-catalyzed acidolysis of a single-cell oil (SCO) containing docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) with caprylic acid (CA) was investigated. The targeted products were structured lipids containing CA residues at the sn-1 and -3 positions and a DHA or DPA residue at the sn-2 position of glycerol. Rhizomucor miehei lipase (RML) and Pseudomonas sp. KWI-56 lipase (PSL) were used as the biocatalysts. When PSL was used > 60 mol% of total SCO fatty acids (FA) were exchanged with CA, with DHA and DPA as well as the other saturated FA being exchanged. The content of the triacylglycerols (TG) containing two CA and one DHA or DPA (number of carbon atoms = 41, i.e., C41) residue was high (36%), and the isomer with the desired configuration (unsaturated FA residue at the sn-2 position) represented 77–78% of C41. In the case of RML, CA content reached only 23 mol% in the TG. A large amount of DHA and DPA residues remained unexchanged with RML, so that the resulting oil was rich in TG species containing two or three DHA or DPA residues (46%). TG C41 amounted to 22%, almost all of which had the desired configuration. This result suggested that the difference in the degree of acidolysis by the two enzymes was due to their different selectivity toward DHA and DPA, as well as the difference in their positional specificities.

6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.

A high-resolution method based on silver ion high-performance liquid chromatography (HPLC) and high-temperature gas chromatography (HTGC) with on-column injection was established for the separation of triacylglycerols (TAG) which contained eicosapentaenoic acid (EPA). A solvent system based on n-hexane, 2-propanol, and acetonitrile in HPLC for the separation of EPA-containing TAG was developed. It was dependent upon the number of EPA molecules and their isomeric distribution on the glycerol backbone. This technique was used to monitor the incorporation of EPA onto specific positions on the glycerol backbone. For quantitative monitoring of the synthesis reaction of the TAG containing EPA, an HTGC analysis was developed. For monitoring of the synthesis of structured TAG containing EPA, transesterification of tricaprylin with EPA ethyl ester in a solvent-free system was performed. The transesterification reaction using various lipases was studied at a different molar ratios of substrates, several initial water activities (aw), and various reaction temperatures.

Pseudomonas lipase was used as catalyst for the transesterification of docosahexaenoic acid ethyl ester with glycerol under low pressure in a solvent free system. The biocatalyst performance was intensified greatly by prior adsorption of lipase on very fine CaCO 3 powder. CaCO 3 particles played the role of a surfactant and of an enzyme transporter to the glycerol-ethyl ester interface. Microscopic analysis of the emulsified reaction mixtures revealed that CaCO 3 particles stabilized the droplets of substrate by adsorption on their surface. The reaction rate, when the immobilized enzyme was used, was five times higher than that of the reaction performed with dissolved free lipase, due to the larger interfacial area available for the enzyme action. The reaction with immobilized enzyme reached equilibrium at 96% consumption of ethyl ester after 8 h of reaction time. In contrast, the reaction with free enzyme was far from equilibrium at only 43% conversion at the same reaction time. The prior immobilization of lipase on CaCO 3 proved to be necessary for the operational stability of the catalyst by protecting the enzyme against inactivation.

Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA) . (g total biomass)-'] and the highest rates of PHA accumulation [(g PHA) (g residual biomass) -1 . h -1 ] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass h -1 ) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through β-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.

Pseudomonas lipase immobilized on CaCO 3 powder was used for the glycerolysis of n −3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO 3 powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.

Cell-free protein synthesis systems enable the direct in vitro expression of proteins from template DNA or RNA. Use of biochemical and bioengineering techniques has greatly improved the yields and productivities of cell-free systems. In some cases, the yields approach the in vivo levels. Moreover, in vitro systems are capable of rapidly providing artificial polypeptides that greatly facilitate protein engineering. Post-translational modification steps in cell-free systems also offer exciting possibilities as reviewed here.

Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties. (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes.

Plasmids carrying stabilizing loci were used in the expression of phospholipase D (PLD) gene fused with pelB signal sequence by a recombinant strain of Escherichia coli BL21 (DE3) using T7 RNA polymerase mediated expression system. By checking the living cell number and the percentage of the plasmid-bearing cells, it was found that the plasmids involving PLD gene were not stable under noninduced conditions and that, after the induction, the number of plasmid-bearing cells were rapidly decreased to almost zero. Then, a biologically stabilizing locus such as par B, ccd, or par was inserted into the plasmids. The newly constructed plasmids were maintained very stably in the recombinant cells until the cells were induced. However, after the induction, almost all the recombinant cells were rapidly killed due to highly toxic PLD. Using the best one of the stabilized PLD-expressing plasmids, PLD production was improved 2-fold.

By using immobilized lipase for production of monoacylglycerol (MAG) by solid-phase glycerolysis of fats and oils, the enzyme could be recovered easily from the reaction mixture and recycled to reduce the cost of the catalyst. Several support materials (CaCO3, CaSO4·2H2O, Ca2P2O7, and Celite) were screened for immobilization of Pseudomonas sp. lipase by adsorption and tested for solid-phase glycerolysis of olive oil. Immobilization made the reuse of enzyme feasible. CaCO3 proved to be the best support: 90% MAG (wt% in the glycerolfree reaction mixture after 72 h of reaction time) was obtained until the fifth use, 80% after the seventh use, and 60% after the tenth use. The same support was found suitable for immobilization of two other bacterial lipases from Chromobacterium viscosum and Pseudomonas pseudoalkali.

An attempt was made to synthesize ricinoleic acid estolides effectively with a bioreactor system containing immobilized lipase. The optimal water content for reaction changed during synthesis of estolide from ricinoleic acid. The water content needs to be controlled to optimize conditions for each reaction stage and to prepare estolide with a high degree of condensation. The estolide synthesis was attempted to confirm if the reaction would have proceeded to the targeted degree of condensation by controlling the reaction mixture under the optimal water content. The result showed that, with the bioreactor, the reaction could proceed rapidly. A repeated batch reaction was possible in the bioreactor. The amount of lipase used can thus be highly reduced compared with discarding it each time. The loading density of enzyme onto the carrier greatly affected enzymatic activity, with a loading level of 60 mg lipase/g carrier producing 60% more estolide per gram of enzyme than a loading level of 120 mg/g. The estolide product synthesized in the bioreactor showed no coloration during the reaction process. This fact confirmed the validity of the proposed method.

Extensive screening for cell-bound lipase-producing filamentous fungi suitable for immobilization in biomass support particles (BSPs) was done. A total of 91 fungi were assayed for their lipolytic activity after cultivation and immobilization in BSPs. In the cultivation, polypepton and oleic acid were used as an organic nitrogen source and a lipase inducer, respectively. Several species, such as Rhizopus species and Rhizomucor miehei, showed higher cell-bound lipolytic activity than that of Rhizopus chinensis, which is known to have considerable high cell-bound lipolytic activity from previous work. For two strains of fungi (Rhizopus stolonifer, Rhizomucor miehei), the cell-bound lipolytic activities of cells immobilized in BSPs did not show strong dependence on polypepton concentration (10–60 g/liter), while the extracellular lipolytic activities in the culture liquid increased with the increase in the polypepton concentration. Thirteen fungi that showed high cell-bound lipolytic activities were examined...

Acid phosphatase isozymes were isolated from wheat germ. Six isozymes were isolated, and one of them was purified to homogeneity. The molecular mass of the purified isozyme was revealed to be 40 kDa (from an SDS-PAGE analysis), or 47 kDa (from a gel filtration analysis), suggesting that the isozyme is a monomer. The N-terminal amino acid sequence of the isozyme was also determined. All of the isozymes showed similar properties; optimum pH, substrate specificities and metal ion inhibition. However, three isozymes were able to hydrolyze phosphotyrosine, but the others were not. Mouse antiserum raised against the purified isozyme could react with other partially purified isozymes except for one isozyme, suggesting that at least five isozymes isolated in this study immunochemically have common epitopes.

In order to increase the productivity of a cell-free protein synthesis system using wheat-germ extract, the extract was highly condensed by polyethylene glycol precipitation, which greatly increased both the final protein concentration and the initial synthesis rate of translation. However, the translational reaction using the condensed extract ceased earlier than the reaction using the uncondensed extract did, because ribonuclease and phosphatase activities, which destroyed mRNA and lessened the ATP level, respectively, were also condensed. Adding Cu2+ to the condensed translational system was found to reduce these activities resulting in a prolonged reaction with a high initial rate. Finally, 120 μg of dihydrofolate reductase protein from Escherichia coli was obtained in 1 ml of reaction mixture in 3 h. In addition, the initial protein synthesis rate was 70 μg ml−1 h−1, which was about 10 times higher than that of a conventional uncondensed extract.

Alcaligenes latus, a growth-associated PHB producer, was cultivated by a pH-stat modal fed-batch culture technique to attain high PHB productivity. Both sucrose solution and inorganic medium were fed in conjunction with the supply of ammonia solution which serves as a nitrogen source and as a means of pH control. Compositions of the inorganic medium were formulated by elemental analysis of A. latus cell mass. The effect on inoculum size was examined to reduce culture time. High concentrations of cell (142 g/L) and PHB (68.4 g/L) were obtained in a short culture time (18 h) with an inoculum size of 13.7 g/L. The PHB content and the PHB productivity at the end of the fed-batch culture were 50% of dry cell weight and 4.0 g PHB/(L . h), respectively.

Continuous production of phospholipase D (PLD) in an air-lift reactor system was investigated using growing cells of Streptomyces lydicus D-121 immobilized within cross-linked chitosan beads as porous supports. In a continuous culture with the immobilized growing cells, both the activity and productivity were highest at a dilution rate of 0.330 h −1 which was higher than the maximum specific growth rate of the free cells (0.247 h −1 ). The highest PLD activity was obtained at pH 6.0–6.5. The average activity was 12.7 U/ml during 120–384 h in a continuous culture with immobilized cells, while the productivity of the continuous culture (4.19 × 10 3 U/ l ·h) was about 3.3–3.6-fold higher than that of the batch culture (1.16 × 10 3 U/ l ·h) and the repeated-batch culture in the reactor (1.27 × 10 3 U/ l ·h), and about 3-fold higher than that in the case of continuous culture with free cells (1.36 × 10 3 U/ l ·h).

Several alcohols were examined as substrates for the polyhydroxyalkanoate synthesis by Paracoccus denitrificans. The bacterium synthesized a homopolyester of poly(3-hydroxybutyrate) from ethanol. When n-pentanol was used as growth substrate, homopolyester poly(3-hydroxyvalerate) was synthesized, whereas copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulated during bacterial growth on n-propanol. When alcohols were automatically fed as growth substrates, ethanol, n-propanol, and n-pentanol gave higher polyester content. Although poly(3-hydroxybutyrate) was synthesized from methanol or n-butanol, its content was very low. Under nitrogen-deficient conditions, polyester;content in cells increased, especially with ethanol, n-propanol, and n-pentanol. Using a mixture of two alcohols P. denitrificans could synthesize polyesters with varying relative ratios of 3-hydroxybutyrate to 3-hydroxyvalerate.

The beta-ketothiolase gene (phaA) and acetoacetyl-CoA reductase gene (phaB) were isolated from Paracoccus denitrificans. Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40,744 Da and of 242 amino acids with a molecular mass of 25,614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4-74.0% for the phaA gene product and 47.6-80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli. In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA-phaB, in P. denitrificans. NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.

The phospholipase D (PLD)-encoding gene of Streptomyces antibioticus fused with the pectate lyase B (PelB) signal sequence was expressed in recombinant Escherichia coli under the control of the T7 lac promotor. Most of the PLD activity was detected in the culture supernatant. The N-terminal amino acid sequence of the recombinant PLD was identical to that of the wild-type PLD, suggesting that the processing of the PelB signal peptide occurred correctly and that the PLD produced by E. coli was identical to the wild-type enzyme. The production of PLD was improved by genetic and fermentation techniques. About 3 mg of PLD/ l medium was obtained under optimal conditions, comparable to the amount of PLD produced by S. antibioticus . The results of analysis of the distributions of intracellular marker enzymes suggested that neither cell lysis nor periplasmic disruption was the cause of the secretion of PLD.

Estolide has numerous industrial applications, and can be synthesized from ricinoleic acid by heating at 220°C. Due to the high reaction temperature involved, this process leads to product coloring and ordor problems, thus limiting the application of this compound to food processing.Study was thus made to determine whether immobilized lipase could be uaed in the synthesis of estolide. The results obtained are as follows.1) Estolide could be synthesized at 40°C by the catalytic activity of immobilized lipase.2) This activity of immobilized lipase was made five times that of free lipase by co-immobilization with lecithin.3) The condensation rate was made high by the addition of zeolite to remove water generated in the reaction.4) Colorless estolide with a high condensation rate was synthesized using a bioreactor system consisting of a packed bed bioreactor and a dehydration column.

Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato-dextrose-agar medium was studied aiming at starting its L(+)-lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar-on-bottom or agar-on-top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar-on-top mode gave larger amount of spores than in an agar-on-bottom mode at 30°C for 7-day cultivation. Scale-up of the agar plate culture from 26.4 to 292 cm2 was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m3 industrial submerged fermentation started directly from 2 × 105 spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the “full-scale industrial submerged fermentation started directly from spore inoculation omitting pre-culture” has been discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:876–881, 2013