• KSBB Journal
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• v.16 no.6
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• pp.562-567
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• 2001

Aspergillus niger LK harboring the enantioselective epoxide hydrolase (EHase) activity was isolated, and enantioselectivity of EHase was tested for various racemic aromatic epoxides. The gene encoding epoxide hydrolase was cloned from cDNA library generated by reverse transcriptase-polymerase chain reaction of the isolated total mRNA. Sequence analysis showed that the cloned gene encodes 398 amino acids with a deduced molecular mass of 44.5 kDa. Database comparison of the amino acid sequence reveals that it is similar to fungal EHase, whereas the sequence identity with bacterial EHase is very low. Recombinant expression of the cloned EHase in Escherichia coli BL21 yielded an active EHases, which can offer a potential biocatalyst for the production of chiral epoxides.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.55-60
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• 1998

For an ideal extractive bioconversion in aqueous two-phase systems, the product has to be preferentially partitioned into the phase opposite to the one in which the biocatalyst is located. Partitioning behaviors of Lactobacillus helveticus IAM 11090 and lactic acid in aqueous two-phase systems composed a polycation, poly(ethylenimine) (PEI), and an uncharged polymer (hydroxyethyl)cellulose (HEC) were investigated. L. helveticus cells were preferentially partitioned to the HEC-rich top phase while about 85% of lactic acid was partitioned to the PEI-rich bottom phase. These results indicate that extraction of charged, low molecular weight products in an aqueous two-phase systems can be promoted by using an oppositely charged polymer as one of the phase-forming polymer. By the ideal partitioning of the cells and lactic acid, an aqueous PEI/HEC two-phase system can be used as a potential system for the extractive lactic acid fermentation of cheese whey.

• Journal of Life Science
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• v.23 no.10
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• pp.1246-1251
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• 2013

An organic solvent-tolerant lipase of Pseudomonas sp. BCNU 154 that was isolated from wastewater in the industrial complex region had optimal activity at $37^{\circ}C$ and pH 8. This crude extracellular lipase from BCNU 154 exhibited maximum stability in toluene, retaining about 6.01 U/ml (117.53%) activity for 2 h. $Ca^{2+}$, $Mg^{2+}$, $NH_4{^+}$, and $Na^+$ ions and triton X-100 activated the enzymes, whereas $Ba^{2+}$, $Hg^{2+}$, and $Zn^{2+}$ ions inhibited their activity. Pseudomonas sp. BCNU 154 lipase revealed stable activity comparable to that of the commercial immobilized Novozym 435. Thus, this organic solvent-tolerant lipase could have potential as a whole cell biocatalyst in industrial chemical processes without the use of immobilization.

• KSBB Journal
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• v.8 no.4
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• pp.341-350
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• 1993

As an effort to develop an alternative transportation fuel, the production of methanol from methane gas was studied using the resting cells of an obligatory methanotroph, Methylosinus trichosporium OB3b. The reaction was carried out in high concentration phosphate buffer solutions with the flask-grown cells containing the exclusively cytoplasmic methane monooxygenase (sMMO) activity. The methanol accumulation rate was observed to be 79nmo1/mg·min during the initial 4.5hr. Phosphate-dependent inhibition was found for both sMMO and methanol dehydrogenase (MDH) activities, and the inhibition constants were 185mM and 42mM, respectively. The inhibition mode was noncompetitive. Methanol was found to be very inhibitory to the sMMO activity and the inhibition constant (noncompetitive) was 21mM when propylene was used as substrate. The sMO activity in the resting cells was declined very fast and the rate became very high during the methanol production. These results indicate that the use of M. trichosporium OB3b as a biocatalyst for the methanol production is heavily dependent on the stable maintenance of the whole-cell SMO activity as well as the effective alleviation of product inhibition.

• KSBB Journal
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• v.8 no.4
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• pp.313-319
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• 1993

Methyl fructoside was synthesized from sucrose and methanol using an immobilized invertase. The enzyme was covalently bound by glutaraldehyde on porous silica coated with polyethyleneimine to give loading capacity of 120mg of invertase per one gram of dry porous silica and effective activity of 100U per one milligram of bound invertase. Polyethyleneimine coating imparted a hydrophillic character, good activity retention and high loading capacity to the surface of porous silica as well as hydrophillic microenviroment in the vicinity of bound invertase. The immobilized enzyme was formed into an alginate-enclosed silica bead to have enough activity for methyl fructoside synthesis from aqueous methanol-sucrose solution. Using the alginate-enclosed biocatalyst the yield of methyl fructoside was obtained as high as 55.9% from aqueous 30% (v/v) methanol and 0.291mo1/l sucrose with 2U/ml activity at $25^{\circ}C$, pH 4.8.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.6
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• pp.530-537
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• 2006

Recently isolated, Pseudomonas putida SN1 grows on styrene as its sole carbon and energy source through successive oxidation of styrene by styrene monooxygenase (SMO), styrene oxide isomerase (SOI), and phenylacetaldehyde dehydrogenase. For the production of (S)-styrene oxide, two knockout mutants of SN1 were constructed, one lacking SOI and another lacking both SMO and SOI. These mutants were developed into whole-cell biocatalysts by transformation with a multicopy plasmid vector containing SMO genes (styAB) of the SN1. Neither of these self-cloned recombinants could grow on styrene, but both converted styrene into an enantiopure (S)-styrene oxide (e.e. > 99%). Whole-cell SMO activity was higher in the recombinant constructed from the SOI-deleted mutant (130 U/g cdw) than in the other one (35 U/g cdw). However, the SMO activity of the former was about the same as that of the SOI-deleted SN1 possessing a single copy of the styAB gene that was used as host. This indicates that the copy number of styAB genes is not rate-limiting on SMO catalysis by whole-cell SN1.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1067-1076
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• 2016

The gene encoding lipase (Lip98) from Aeromicrobium sp. SCSIO 25071 was cloned and functionally expressed in Escherichia coli. Lip98 amino acid sequence shares the highest (49%) identity to Rhodococcus jostii RHA1 lipase and contains a novel motif (GHSEG), which is different from other clusters in the lipase superfamily. The recombinant lipase was purified to homogeneity with Ni-NTA affinity chromatography. Lip98 showed an apparent molecular mass of 30 kDa on SDS gel. The optimal temperature and pH value for enzymatic activity were recorded at 30℃ and 7.5, respectively. Lip98 exhibited high activity at low temperatures with 35% maximum activity at 0℃ and good stability at temperatures below 35℃. Its calculated activation energy was 4.12 kcal/mol at the low temperature range of 15-30℃. Its activity was slightly affected by some metal ions such as K⁺, Ca²⁺, and Na⁺. The activity of Lip98 was increased by various organic solvents such as DMSO, ethanol, acetone, and hexane with the concentration of 30% (v/v) and retained more than 30% residual activity in neat organic solvent. The unique characteristics of Lip98 imply that it is a promising candidate for industrial application as a nonaqueous biocatalyst and food additive.

• Proceedings of the Membrane Society of Korea Conference
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• 1998.10a
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• pp.15-18
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• 1998

Membrane reactors are systems which combine a chemical reactor with a membrane separation process allowing to carry out simultaneously conversion and product separation. The catalyst can be immobilized on the membrane or simply compartmentalized in a reaction space by the membrane. Membrane reactors are today investigated to produce optically pure isomers and/or resolve racemic mixture of enantiomers. The interest towards these systems is due to the increasing demand of enantiomerically pure compounds to be used in the pharmaceutical, food, and agrochemical industries. In fact, enantiomers can have different biological activities, which often influence the efficacy or toxicity of the compound. On the basis of current literature there are basically two schemes on the use of membrane technology to produce enantiomers. In one case, the membrane itseft is intrinsically enantioselective: the membrane is the chiral system which selectively separates the wanted isomer on the basis of its conformation. In the other, a kinetic resolution using an enantiospecific biocatalyst is combined with a membrane separation process; the membrane separates the product from the substrate on the basis of their relative chemical properties (i.e. solubility). This kind of configuration is widely used to carry out kinetic resolutions of low water soluble substrams in biphasic membrane reactors [Giomo, 1995, 1997; Lopez, 1997]. These are systems where enzyme-loaded membranes promote reactions between two separate phases thanks to the properties of enzymes, such as lipases, to catalyse reactions at the org ic/aqueous interface; the two phases are maintained in contact and separated at the membrane level by operating at appropriate transmembrane pressure. A schematic representation of biphasic membrane reactor is shown in figure 1, while an example of enantiospecific reaction and product separation carried out with these systems is reported in figure 2.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1455-1464
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• 2021

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40℃, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.245-254
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• 2022

Cellulases are a group of biocatalyst enzymes that are capable of degrading cellulosic biomass present in the natural environment and produced by a large number of microorganisms, including bacteria and fungi, etc. In the current study, we isolated, screened and characterized cellulase-producing bacteria from soil. Three cellulose-degrading species were isolated based on clear zone using Congo red stain on carboxymethyl cellulose (CMC) agar plates. These bacterial isolates, named as HB2, HS5 and HS9, were subsequently characterized by morphological and biochemical tests as well as 16S rRNA gene sequencing. Based on 16S rRNA analysis, the bacterial isolates were identified as Bacillus cerus, Bacillus subtilis and Bacillus stratosphericus. Moreover, for maximum cellulase production, different growth parameters were optimized. Maximum optical density for growth was also noted at pH 7.0 for 48 h for all three isolates. Optical density was high for all three isolates using meat extract as a nitrogen source for 48 h. The pH profile of all three strains was quite similar but the maximum enzyme activity was observed at pH 7.0. Maximum cellulase production by all three bacterial isolates was noted when using lactose as a carbon rather than nitrogen and peptone. Further studies are needed for identification of new isolates in this region having maximum cellulolytic activity. Our findings indicate that this enzyme has various potential industrial applications.