Improving the Thermo-Activity and -Stability of Pectate Lyase from Dickeya dadantii DCE-01 for Ramie Degumming

To improve the thermal stability of pectate lyase for ramie degumming, we modified the novel pectate lyase gene (pelG403) derived from the Dickeya dadantii DCE-01 high-efficiency ramie degumming strain by site-directed mutagenesis. Twelve mutants were acquired, wherein a prospective mutant (A129V) showed better enzyme activity and thermal stability. Compared with the wild type (PelG403), the specific enzyme activity and the optimal reaction temperature of A129V in the fermentation broth increased by 20.1%, and 5 °C, respectively. Under the conditions of 55 °C and pH 9.0, the weightlessness rate of ramie raw materials of A129V increased by 6.26%. Therefore, this study successfully improved the enzyme activity and heat resistance of PelG403 in an alkaline environment, which may contribute to the development of enzyme preparations and the elucidation of the mechanism for ramie bio-degumming.

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production

Background Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use. Results Three GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM− 1 s− 1). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h. Conclusions Three recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.

Isolation of Xylanase Producing Strains, Optimization of Fermentation Conditions and Research on Enzymatic Properties

In this study, we successfully isolated a strain of Aspergillus oryzae TR08, which produced xylanase secreted to the outside of the cell productively. The enzyme activity and specific activity in the fermentation broth of this strain reached peak values of 451 IU/mL and 1963 IU/mg after 156 h of fermentation. A single factor experiment was designed, and it was found that the strain was adjusted to the initial pH of the fermentation broth to 7.5 in a shaker at 180 rpm and 32 °C. After 156 h of fermentation, the enzyme activity reached a maximum of 1264 IU/mL. The optimal reaction temperature and pH value of the xylanase were 55 °C and 7.5, respectively, and it had excellent acid and alkali resistance and a wide pH activity range. The xylanase was increased the catalytic activity by 15% in 0.25 mM Fe3+, and the biological activity of the enzyme was not affected in the sodium dodecyl sulfate environment.

Characterization of Three GADs from Bacillus spp. for Efficient γ-Aminobutyric Acid Production

BackgroundGamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use.ResultsThree GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM− 1·s− 1). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 8 times, the cells produced GABA with an average molar conversion rate of 97% within 8 h.ConclusionsThree recombinant GADs from Bacillus strains have been conducted functional identification. And engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.

Cloning and Expression of Levansucrase Gene of Bacillus velezensis BM-2 and Enzymatic Synthesis of Levan

Levan is a versatile and valuable fructose homopolymer, and a few bacterial strains have been found to produce levan. Although levan products have numerous specific functions, their application and promotion were limited by the production capacity and production cost. Bacillus velezensis BM-2 is a levan-synthesizing strain, but its levan production is too low to apply. In this study, the levansucrase gene of B. velezensis BM-2 was cloned to plasmid pET-32a-Acma-zz, and the recombinant plasmids were transferred to Escherichia coli BL21. A transformed clone was selected to express and secrete the fusion enzymes with an Acma-tag efficiently. The expressed products were further purified by a self-developed separating material called bacterial enhancer matrix (BEM) particles. The purification efficiency was 93.4%, with a specific activity of 16.589 U/mL protein. The enzymatic reaction results indicated that the optimal reaction temperature is 50 °C, the optimal pH of the acetate buffer is 5.6, and the buffer system greatly influenced the enzyme activity. The enzyme activity was enhanced to 130% in the presence of 5 mM Ca2+, K+, Zn2+, and Mn2+, whereas it was almost abolished in the case of Cu2+ and Fe3+. The values of Km, kcat, and kcat/Km were 17.41 mM, 376.83 s−1, and 21.64 mM−1s−1, respectively. The enzyme amount of 20 U/g sucrose was added to the system containing 400 g/L sucrose, and the levan products with a concentration of 120 g/L reached after an incubation of 18 h, which was 8 times that of the yield before optimization. The results of molecular docking analysis indicated that the Asp86 might act as a nucleophilic catalytic residue for sucrose, Arg246 and Asp247 act as transition state stabilizer of transfructosylation, and Glu340 and Arg306 were recognized as general acid donors. They formed the catalytic-groups triad. The unique properties and catalytic activity of the levansucrase suggest that it deserves further research and might have good industrial application prospects.

Directed evolution and immobilization of new lipase Lip 906

In this experimental study, a new lipase named Lip 906 was screened out from a metagenomic library in the laboratory. To improve the stability of the enzyme and develop and apply it as soon as possible, we adopted directed evolution and immobilization methods. A random mutation library was constructed by error-prone PCR and finally, a mutant lipase Lip 5-D with increased enzyme activity was screened out and immobilized. The activity of the mutant enzyme Lip 5-D was improved by 4 times compared with the wild-type lipase Lip 906. The optimal reaction temperature rose by 4 °C, and by 3 °C after immobilization. The optimal reaction pH increased from 7.8 to 7.5. Both temperature stability and pH stability were improved. The mutant enzyme Lip 5-D can maintain about 70% of the relative activity after incubation at 65 °C for 2 h, and it can keep 60% at pH 3-10. Error-prone PCR and immobilization improve the catalytic activity and stability of the enzyme, and promote its development and application in many industries.

One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4–10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4-10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4-10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

Characterization of a New Bifunctional and Cold-Adapted Polysaccharide Lyase (PL) Family 7 Alginate Lyase from Flavobacterium sp.

Alginate oligosaccharides produced by enzymatic degradation show versatile physiological functions and biological activities. In this study, a new alginate lyase encoding gene alyS02 from Flavobacterium sp. S02 was recombinantly expressed at a high level in Yarrowia lipolytica, with the highest extracellular activity in the supernatant reaching 36.8 ± 2.1 U/mL. AlyS02 was classified in the polysaccharide lyase (PL) family 7. The optimal reaction temperature and pH of this enzyme were 30 °C and 7.6, respectively, indicating that AlyS02 is a cold-adapted enzyme. Interestingly, AlyS02 contained more than 90% enzyme activity at 25 °C, higher than other cold-adapted enzymes. Moreover, AlyS02 is a bifunctional alginate lyase that degrades both polyG and polyM, producing di- and trisaccharides from alginate. These findings suggest that AlyS02 would be a potent tool for the industrial applications.