Yihan Liu

Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations

This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest kcat/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The kcat/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.

High-yield phosphatidylserine production via yeast surface display of phospholipase D from Streptomyces chromofuscus on Pichia pastoris.

The gene encoding phospholipase D (PLD) from Streptomyces chromofuscus was displayed on the cell surface of Pichia pastoris GS115/pKFS-pldh using a Flo1p anchor attachment signal sequence (FS anchor). The displayed PLD (dPLD) showed maximum enzymatic activity at pH 6.0 and 55 °C and was stable within a broad range of temperatures (20-65 °C) and pHs (pH 4.0-11.0). In addition, the thermostability, acid stability and organic solvent tolerance of the dPLD were significantly enhanced compared with the secreted PLD (sPLD) from S. chromofuscus. Use of dPLD for conversion of phosphatidylcholine (PC) and l-serine to phosphatidylserine (PS) showed that 67.5% of PC was converted into PS at the optimum conditions. Moreover, the conversion rate of PS remained above 50% after 7 repeated batch cycles. Thus, P. pastoris GS115/pKFS-pldh shows the potential for viable industrial production of PS.

Extracellular expression of pullulanase from Bacillus naganoensis in Escherichia coli

The pullulanase encoding gene from Bacillus naganoensis was successfully overexpressed in Escherichia coli both intracellularly and extracellularly using expression vector pET22b (+). The distribution of recombinant protein was significantly affected by temperature and carbon and nitrogen sources. The highest levels of extracellular and intracellular production of the target protein were observed at 25 and 20 °C, respectively. The addition of maltose, dextrin, pullulan, and soluble starch to the culture medium caused significant increases in the extracellular yield of pullulanase, while glucose strongly inhibited pullulanase production. The results show that the optimal conditions for maximum yield of extracellular pullulanase required high levels of carbon source and a limited nitrogen supply, while low concentrations of carbon and nitrogen source favored intracellular pullulanase expression. High concentrations of nitrogen source strongly inhibited the production of pullulanase.

High-level expression of the Streptomyces mobaraense CICC 11018 transglutaminase in Corynebacterium glutamicum ATCC 13032

The transglutaminase (MTG) secreted by Streptomyces mobaraense is a valuable enzyme for the food industry. To optimize the expression system for MTG heterologous expression in Corynebacteriumglutamicum ATCC 13032, we took advantage of the recently identified efficient secretion signal peptide ΔS0949 and the tac-M promoter, a modified version of tac promoter. MTG activity assay showed that the recombinant C. glutamicum ATCC 13032 harboring plasmid pXMJ19-Spromtg which encodes the pro-MTG fused to the signal peptide ΔS0949 secreted pro-MTG to the culture broth with MTG activity of 5.2 U/mL. However, the recombinant strain pXMJ19-MSpromtg/13032 containing both the signal peptide ΔS0949 and the tac-M promoter accumulated pro-MTG up to a level of 6.7 U/mL, that resulted in an increase of 28.8% compared with the pXMJ19-Spromtg/13032 strain. Semi-quantitative RT-PCR demonstrated that transcriptional level of mtg driven by the tac-M promoter was 1.5-fold higher than that driven by the tac promoter, suggesting that, for the expression system used in this study, level of mtg transcription was a limiting factor for high-level MTG production in C. glutamicum ATCC 13032. Thus, further improvement of MTG production in the heterologous host C. glutamicum could be achieved by the use of more powerful promoters.

Improvement in thermostability of an alkaline lipase I from Penicillium cyclopium by directed evolution

Lipases have been used in a wide range of applications, and thermostable lipases are highly required. Lipase I (PCL) from Penicillium cyclopium with alkali stability showed poor thermostability, resulting in a limited application in industrial processes with high temperatures. In this study, to expand the functionality of PCL, the thermostability of PCL was enhanced through direct evolution with error-prone PCR. Using a high-throughput activity assay, the double mutant PCL (L41P/G47I) with improved thermostability was selected, and then the single mutants L41P and G47I were constructed via site-directed mutagenesis. The mutant PCLs exhibited an optimum temperature 5 °C higher than the wild-type (WT). WT showed no activity with incubation at 40 °C for 90 min, whereas L41P, G47I, and L41P/G47I displayed 11%, 44%, and 23% of their maximal activity after incubation at 45 °C for 60 min. Meanwhile, the half-life at 45 °C of L41P, G47I, and L41P/G47I was 7-, 13-, and 9-fold higher, respectively, than that of WT. Moreover, G47I, L41P/G47, L41P, and WT were successfully applied in the synthesis of ethyl propionate with conversions of 45%, 27%, 23%, and 8%. The studies of circular dichroism spectroscopy and fluorescence spectroscopy indicated the lower flexibility and higher compactness of the structure of the mutant PCLs as compared with WT. Combining these results and analysis of the three-dimensional structure modeling, the mechanism responsible for the improvement of the thermostability was elucidated, which demonstrated that Leu41Pro and Gly47Ile could stabilize the structure of PCL through hydrophilic/hydrophobic interactions, proline substitution, and helix propensity.

Cloning, expression and characterisation of phospholipase B from Saccharomyces cerevisiae and its application in the synthesis of l-alpha-glycerylphosphorylcholine and peanut oil degumming

ABSTRACT l-alpha-glycerylphosphorylcholine (GPC) has been shown to enhance cognitive performance. Meanwhile, vegetable oils must be refined to remove the impurities for them to be edible. Phospholipase B (PLB), having the ability of hydrolyzing both the sn-1 and sn-2 acyl ester bonds of phospholipids, can produce GPC using PC as substrate and transform the non-hydratable phospholipids into their hydratable forms. The Saccharomyces cerevisiae plb gene, which encodes PLB, was cloned and expressed in Pichia pastoris GS115 to produce recombinant PLB (rPLB). Fermentation optimisation yielded rPLB activity levels as high as 1723 U/mL. rPLB demonstrated maximum enzymatic activity at 40 °C and pH 5.5 and was stable at temperatures between 30 and 40 °C and pH values between 5.0 and 6.0. rPLB synthesised GPC with a conversion rate of 17% (w/w) and exhibited high degumming activity towards peanut oil, decreasing the phosphorus content from 91.8 to 3.7 mg/kg within 3 h. This study describes a candidate phospholipase for potential applications involving the modification of phospholipids and vegetable oil degumming.

Optimization of Sample Preparation for the Metabolomics of Bacillus licheniformis by GC-MS

Metabolomic has become an important method in microbiology study. Sample preparation affects the quality of final metabolomics analysis strongly. There is no universal preparation method that can suit the metabolomics study of all kinds of microorganism, because of their various cell structure. In this study, a suitable sample preparation method for the metabolomics study of Bacillus licheniformis was explored and optimized. The main steps in metabolite sample preparation include quenching, estimation of leakage, and metabolite extraction. The result indicated that 60 % methanol, 0.9 % ammonium carbonate buffer was an appropriate quenching solution for Bacillus licheniformis by measuring intracellular metabolites, energy charge, and intracellular metabolites. Among the four different extraction methods (cold pure methanol, PM; cold methanol/water (70:30 v/v), MW; acetonitrile/methanol/water, (2:2:1 v/v/v), AMW; or acetonitrile/water (1:1 v/v), AW), MW was superior to others on the intracellular metabolites, which could effectively extract more intracellular metabolites. The results imply that the optimized preparation method for Bacillus licheniformis is critical for a reliable and accurate analysis of metabolome.

A novel process for phosphatidylserine production using a Pichia pastoris whole-cell biocatalyst with overexpression of phospholipase D from Streptomyces halstedii in a purely aqueous system

This work was aimed to develop a novel phosphatidylserine (PS) production process for the food industry. The pldsh gene, encoding phospholipase D from Streptomyces halstedii (PLDsh) was cloned, and the codon optimized pldmsh gene was freely expressed by Pichia pastoris GS115 and successfully overexpressed on the cell surface of P. pastoris GS115 as displayed PLDMsh (dPLDMsh) - a whole-cell biocatalyst for PS synthesis from phosphatidylcholine and l-serine. dPLDMsh was stable over a broad range of temperatures (20-60 °C) and pH values (4.0-8.0), indicating significant improvement in stability compared with its free counterpart expressed by P. pastoris GS115. Under the optimum conditions, the conversion yield of PS was 53%, and the relative yield remained above 40% after 4 repeated batch cycles of dPLDMsh catalysis in an aqueous system. Thus, dPLDMsh and the associated reaction system provided a novel strategy for efficient PS production for the food industry.

Development of a Pichia pastoris whole-cell biocatalyst with overexpression of mutant lipase I PCLG47I from Penicillium cyclopium for biodiesel production

Penicillium cyclopium lipase I (PCL) is a thermolabile triacylglycerol lipase with very low activity against monoacylglycerols, and there have been no reports on the transesterification of oil to produce biodiesel. A mutant PCLG47I with an improved thermostability was previously obtained through replacing Gly47 with Ile in PCL. In this study, a novel Pichia pastoris whole-cell biocatalyst (WCB) with overexpression of PCLG47I was constructed and characterized for biodiesel production from soybean oil. The optimum conditions for biodiesel preparation were 1 g soybean oil, 1 : 2 initial oil/methanol molar ratio with 3 times methanol addition of 1 : 0.75 oil/methanol molar ratio at 4 h intervals, 7% water content, 400 U lipase, temperature of 25 °C, and reaction time of 20 h. Under the optimum conditions, the FAME yield reached 60.7% and remained 47.3% after 4 batch cycles, and no glycerol was generated as a byproduct. These findings indicated that this WCB is a promising biocatalyst for biodiesel production in a relatively cost-effective manner. Additionally, the resulting enzymatic process may provide a potential method for biodiesel production at an industrial scale.

Metagenomic Profiling of the Bacterial Community Changes from Koji to Mash Stage in the Brewing of Soy Sauce

The improvement of soy sauce fermentation is restricted by the insufficient information on bacterial community. In this study, bacterial communities in the koji and mash stage were compared based on next-generation sequencing technology. A total of 29 genera were identified in the koji stage, while 34 in the mash stage. After koji stage, 7 genera disappeared and 12 new genera appeared in the mash stage. The dominant bacteria were Kurthia, Weissella and Staphylococcus in the koji stage and Staphylococcus, Kurthia, Enterococcus and Leuconostoc in the mash stage. The results provided insights into the microbial communities involved in soy sauce fermentation.