The alginate lyase from Isoptericola halotolerans CGMCC 5336 as a new tool for the production of alginate oligosaccharides with guluronic acid as reducing end.

Alginate oligosaccharides are depolymerization products of alginate by enzymatic degradation or physicochemical treatments. Alginate lyase has received great interests due to its use in oligosaccharide preparation. The substrate specificity of alginate lyase directly determines the type and degree of polymerization of alginate oligosaccharides. In this paper, the degradation products of Alginate lyase from Isoptericola halotolerans CGMCC 5336 were size-fractionated by gel filtration chromatography and the fractions were characterized by TLC, HPLC, MS and 1H NMR spectroscopy. The Mw values for these fractions were 352, 528, 704 Da which represented di-, tri-, and tetrasaccharide fragments, respectively. The 1H NMR revealed that the non-reducing ends of these oligosaccharides were 4-deoxy-L-erythro-hex-4-enepyranosyluronate and the reducing ends of these oligosaccharides were all guluronic acid. Therefore, it is believed that the alginate lyase produced by Isoptericola halotolerans CGMCC 5336 was capable of performing ß elimination on guluronic acid residue so that low molecular weight oligosaccharides with guluronic acid on the reducing end could be obtained in this way.

Synthetic oligosaccharides can replace animal-sourced low-molecular weight heparins.

Low-molecular weight heparin (LMWH) is used clinically to treat clotting disorders. As an animal-sourced product, LMWH is a highly heterogeneous mixture, and its anticoagulant activity is not fully reversible by protamine. Furthermore, the reliability of the LMWH supply chain is a concern for regulatory agencies. We demonstrate the synthesis of heparin dodecasaccharides (12-mers) at the gram scale. In vitro experiments demonstrate that the anticoagulant activity of the 12-mers could be reversed using protamine. One of these, labeled as 12-mer-1, reduced the size of blood clots in the mouse model of deep vein thrombosis and attenuated circulating procoagulant markers in the mouse model of sickle cell disease. An ex vivo experiment demonstrates that the anticoagulant activity of 12-mer-1 could be reversed by protamine. 12-mer-1 was also examined in a nonhuman primate model to determine its pharmacodynamic parameters. A 7-day toxicity study in a rat model showed no toxic effects. The data suggest that a synthetic homogeneous oligosaccharide can replace animal-sourced LMWHs.

Nitrile-hydrolyzing enzyme from Meyerozyma guilliermondii and its potential in biosynthesis of 3-hydroxypropionic acid.

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag+, Pb2+ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

A novel alkaline surfactant-stable keratinase with superior feather-degrading potential based on library screening strategy.

A novel keratinase was mined and expressed in Escherichia coli BL21 (DE3) via function-driven screening with fosmid library. The catalytic properties of purified keratinase were investigated in detail following enzyme purification. The recombinant keratinase was purified to homogeneity with an estimated molecular weight of 26kDa using nickel affinity chromatography, of which the optimal reaction pH and temperature were 10.0 and 55°C, respectively. It could remain stable at pH 5.0-12.0 and 40-60°C. Metal ions such as Ca2+, Mn2+, Ag+, Na+, Mg2+, Li+, Sn2+ (1mM) displayed positive influence on keratinase, and particularly, Ca2+ exhibited remarkable improvement effect by 2.6 folds. It was strongly inhibited by PMSF as a protease inhibitor. On the contrary, it could be obviously activated by various surfactants, such as Tween 40 and Triton X-114. The recombinant keratinase showed high specificity towards casein, soluble keratin, BSA, and wool. The keratinase could efficiently degrade the feathers, which demonstrated its applicable potential for biodegradation of keratin wastes and regeneration of soluble protein.

Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798.

Dehairing is a high pollution process in leather industry. Conventionally, the lime-sulfide mediated chemical process for dehairing would lead to the discharge of pollutants and corrosion of industrial equipment. Concerning these problems, keratinase has become a promising candidate for dehairing process in recent years. In this study, a keratinase-producing bacterium was isolated from sheepfold soil and identified as Brevibacillus parabrevis CGMCC 10798 based on the biochemical characteristics and molecular identification. The keratinase was purified to electrophoretic homogeneity with 17.19% of recovery, 13.18 folds of purification and an estimated molecular weight of 28kDa. The enzyme exhibited high keratinase activity and no collagenase activity. Besides, the keratinase showed optimal activity at 60°C and pH 8.0. The enzyme activity could be significantly increased in the presence of Na+ and Ca2+. And it was inhibited by EDTA, and PMSF, which indicated that the keratinase belongs to serine-metallo protease. The enzyme could remain stable in the presence of surfactants. Especially, 5mM Tween 40 and Triton 100 could improve the activity by 11% and 30%, respectively. Moreover, B. parabrevis keratinase could completely dehair goat wool within 7h, which indicated its application potential in leather industry.

Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum.

Corynebacterium crenatum SYPA5-5, an L-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits L-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by L-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (L-citrulline and L-ornithine) and decreased production of L-arginine. Genetic and enzymatic levels analysis involved in L-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from L-citrulline to L-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased L-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the L-arginine productivity improved about 63.6%. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM L-arginine with the productivity of 5.42 mM h(-1). These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward L-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for L-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct L-arginine high-producing C. crenatum.

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate.

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.

Effect of cofactor folate on the growth of Corynebacterium glutamicum SYPS-062 and L-serine accumulation.

The direct fermentative production of L-serine from sugar has attracted increasing attention. Corynebacterium glutamicum SYPS-062 can directly convert sugar to L-serine. In this study, the effects of exogenous and endogenous regulation of cofactor folate on C. glutamicum SYPS-062 growth and L-serine accumulation were investigated. For exogenous regulation, the inhibitor (sulfamethoxazole) or precursor (p-aminobenzoate) of folate biosynthesis was added to the medium, respectively. For endogenous regulation, the gene (pabAB) that encodes the key enzyme of folate biosynthesis was knocked out or overexpressed to obtain the recombinant C. glutamicum SYPS-062 ΔpabAB and SYPS-062(pJC-tac-pabAB), respectively. The results indicated that decreased levels of cofactor folate supported L-serine accumulation, whereas increased levels of cofactor folate aided in cell growth of C. glutamicum SYPS-062. Thus, this study not only elucidated the role of folate in C. glutamicum SYPS-062 growth and L-serine accumulation, but also provided a novel and convenient approach to regulate folate biosynthesis in C. glutamicum.

Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase.

Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336.

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0-8.0) and temperatures below 50 °C. Metal ions including Na(+), Mg(2+), Mn(2+), and Ca(2+) notably increased the activity of the enzyme. With sodium alginate as the substrate, the Km and Vmax were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.

The effect of a LYSE exporter overexpression on L-arginine production in Corynebacterium crenatum.

Corynebacterium crenatum SYPA is an aerobic and industrial L-arginine producer. A lysE deletion mutant was constructed searching a natural function of this in C. crenatum SYPA. Tracking determination of intracellular and extracellular L-arginine in the minimum culture CGXII showed that LysE did play an important role in transporting L-arginine from intracellular to extracellular, without which the accumulation of intracellular L-arginine of C. crenatum SYPA could reach an extremely high concentration. Nevertheless, the lower intracellular concentration of L-arginine could decrease the feedback inhibition in the biosynthesis of L-arginine. To improve the yield from excretion, we attempted to achieve overexpress in the L-arginine transporter in C. crenatum SYPA. Thus, the heterologous and homologous expressions of EcargO and CclysE, respectively, were carried out by means of the shuttle plasmid pJCtac in C. crenatum SYPA. It was found that L-arginine production was improved by 13.6 % because of the overexpression of lysE in the strain (SYPA/pJCE) contrast to the wild-type strain and reached 35.9 g/L, whereas L-arginine production of the strain SYPA/pJCO was scarcely changed. Consequently, we succeeded in obtaining a mutant form of LysE which has an effect to decrease the intracellular concentration of L-arginine in an L-arginine producer of C. crenatum SYPA and found that the production of L-arginine by C. crenatum carrying lysE was improved. These results show the importance of the factor (lysE) involved in the excretion of L-arginine on its over-production in C. crenatum.

The role of ARGR repressor regulation on L-arginine production in Corynebacterium crenatum.

Corynebacterium crenatum SYA is an aerobic, Gram-positive, non-sporulating coryneform bacterium, and the mutant C. crenatum strain SYPA 5-5 can produce 30 g/l L-arginine under optimal culture conditions. In this study, the evolution of the cluster argCJBDFRGH (argC~H) involved in arginine biosynthesis in C. crenatum SYA, and SYPA 5-5 was investigated. Compared to the argR of its wild type C. crenatum SYA, a nucleotide substitution (C→T) within the argR gene of the mutant C. crenatum strain SYPA 5-5 was found. The inactivation of ARGR resulted in increased enzyme activities involved in L-arginine biosynthesis and increased L-arginine production in C. crenatum. In contrast, constructing an overexpressing argR C. crenatum/pTR, a complete and functional ARGR decreased the expression of enzymes, depressed transcriptional level of the argC~H cluster, and reduced the production of L-arginine in C. crenatum. It was thus evident that the inactivation of an ARGR suppressor could relieve a bottleneck in downstream steps of the L-arginine biosynthetic pathway, providing a good strategy for improving L-arginine production.

[Expression, purification and characterization of K5 lyase in Escherichia coli].

K5 polysaccharide of high molecular weight (HLW) can be splitted into low molecular weight (LMW) K5 polysaccharide by K5 lyase which can be used as the substrate of partial synthesis low molecular heparin sulfate (HS). To prepare recombinant K5 lyase (Elma) and analyze its biological activity. The gene of Elma was cloned by PCR amplification and was ligated with pET-28a. Then the recombinant expression vector pET-28a-Elma was transformed into Escherichia coli BL21 (DE3). After induction with 0.2 mmol/L IPTG at 16 degrees C for 5 h, Elma was successfully expressed, SDS-PAGE analysis demonstrated that the enzyme constituted more than 30% of the total cell proteins. After Ni(2+)-NTA affinity and G-75 chromatography, the purity of enzyme was more than 95%. Enzymatic activity was determined according to the change of absorbance at 232 nm per ml of the sample. The reduction of the polysaccharide molecular weight could be detected by PAGE electrophoresis. Elma can partially split HA and HS. Its optimal reatcion temperature is 37 degrees C and the optimal reaction pH is 7.0.

Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of (L) -arginine production.

The genes involved in L: -arginine biosynthesis in Corynebacterium crenatum are organized as the argCJBDFRGH cluster like in Corynebacterium glutamicum. However, the argC~H cluster of the C. crenatum SYPA 5-5, which is an industrialized L: -arginine producer, had a lethal mutation occurring in the ArgR repressor encoding gene. The argC~H cluster with an inactive argR was overexpressed in E. coli and C. crenatum. In the recombinant E. coli JM109 enzyme activities were increased, and more L: -arginine was found in the supernatants from L: -glutamine. When the argC~H cluster was overexpressed in C. crenatum under its native promoter Parg, L: -arginine production was increased by 24.9%, but the presence of the recombinant plasmid pJC-9039 had a negative effect on cell growth. Surprisingly, the DO value of the recombinant strain dropped gently and stayed at a lower level from 24 h to the end of fermentation. The results demonstrated an increasing utilization of oxygen and the distinct enhancement of unit cell L: -arginine yields with the cluster argC~H-bearing in C. crenatum SYPA-9039. This study provides a kind of Corynebacteria with improved L: -arginine-producing ability and an efficient elevation for producing amino acid. Moreover, the promoter Parg would be used as a valid promoter to express objective genes for metabolic engineering in Corynebacteria.

Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum.

Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-L-glutamate (NAG) to N-acety-L-glutamy-L-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the L-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the L-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I0.5 (R) 50-60 fold, and the G287D and R209A mutants could increase the I0.5 (R) 30-40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition.

Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production.

N-acetyl-L-glutamate kinase (EC 2.7.2.8) is first committed in the specific L-arginine pathway of Corynebacterium sp. A limited increase of L-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that L-arginine feedback inhibition plays an influence on the L-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I0.5R efficiently. Thereby, the multi-mutated NAGKM3 (including E19R/H26E/H268D) was generated and its I0.5R of L-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust L-arginine producer, the engineered strains SYPA-CCBM3 were constructed. Introducing the argBM3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The L-arginine synthesis was largely enhanced due to the overexpression of the argBM3, which is resistant to feedback resistant by L-arginine. Thus L-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the L-arginine metabolic pathway by deregulating the key enzyme of the pathway.

[High-level expression of fusion protein GGH in Pichia pastoris GS115 by constructing a double plasmid co-expression system].

In order to make a large-scale preparation of(GLP-1A2G)2-HAS (GGH), the double-plamid pPICZalphaB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZalphaB-ggh, which was transformed into P. pastoris GS 115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS 115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 degrees C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS 115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.

[Cloning, expression and characterization of N-acetylornithine aminotransferase from Corynebacterium crenatum and its effects on L-arginine fermentation].

N-Acetylornithine aminotransferase (EC 2.6.1.11, ACOAT) catalyzes the conversion of N-acetylglutamic semialdehyde to N-acetylornithine, the forth step involved in the L-arginine biosynthetic pathways. We studied the enzyme properties to set up reliable theoretical basis for the arginine fermentation optimization. ACOAT encoding gene argD was cloned from an industrial L-arginine producer Corynebacterium crenatum SYPA 5-5. Analysis of argD sequences revealed that only one ORF existed, which coded a peptide of 390 amino acids with a calculated molecular weight of 41.0 kDa. The argD gene from C. crenatum SYPA 5-5 was expressed both in Escherichia coli BL21 and C. crenatum SYPA. Then ACOAT was purified by Ni-NTA affinity chromatography and its specific enzyme activity was 108.2 U/g. Subsequently, the expression plasmid pJCtac-CcargD was transformed into C. crenatum SYPA and the specific activity of ACOAT was improved evidently in the recombinant C. crenatum CCD. Further fermentative character of CCD1 was also analyzed. The results showed that the L-arginine producing ability of the recombinant strain was 39.7 g/L improved by 14.7%.

Isolation, identification, and culture optimization of a novel glycinonitrile-hydrolyzing fungus-Fusarium oxysporum H3.

Microbial transformation of glycinonitrile into glycine by nitrile hydrolase is of considerable interest to green chemistry. A novel fungus with high nitrile hydrolase was newly isolated from soil samples and identified as Fusarium oxysporum H3 through 18S ribosomal DNA, 28S ribosomal DNA, and the internal transcribed spacer sequence analysis, together with morphology characteristics. After primary optimization of culture conditions including pH, temperature, carbon/nitrogen sources, inducers, and metal ions, the enzyme activity was greatly increased from 326 to 4,313 U/L. The preferred carbon/nitrogen sources, inducer, and metal ions were glucose and yeast extract, caprolactam, and Cu(2+), Mn(2+), and Fe(2+), respectively. The maximum enzyme formation was obtained when F. oxysporum H3 was cultivated at 30 °C for 54 h with the initial pH of 7.2. There is scanty report about the optimization of nitrile hydrolase production from nitrile-converting fungus.

Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum.

Ornithine acetyltransferase (EC 2.3.1.35; OATase) gene (argJ) from the L-arginine-producing mutant Corynebacterium crenatum SYPA5-5 was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). Analysis of the argJ sequence revealed that the argJ coded a polypeptide of 388 amino acids with a calculated molecular weight of 39.7 kDa. In this study, the function of the OATase (argJ) of C. crenatum SYPA5-5 has been identified as a conserved ATML sequence for the autolysis of the protein to α- and ß-subunits. When the argJ regions corresponding to the α- and ß-subunits were cloned and expressed separately in E. coli BL21, OATase activities were abolished. At the same time, a functional study revealed that OATase from C. crenatum SYPA5-5 was a bifunctional enzyme with the functions of acetylglutamate synthase (EC 2.3.1.1, NAGS) and acetylornithine deacetylase (EC 3.5.1.16, AOase) activities. In order to investigate the effects of the overexpression of the argJ gene on L: -arginine production, the argJ gene was inserted into pJCtac to yield the recombinant shuttle plasmid pJCtac-argJ and then transformed into C. crenatum SYPA5-5. The results showed that the engineered strains could not only express more OATase (90.9%) but also increase the production of L: -arginine significantly (16.8%).