Yutuo Wei

A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR.

A rapid and efficient method to perform site-directed mutagenesis based on an improved version of overlap extension by polymerase chain reaction (OE-PCR) is demonstrated in this paper. For this method, which we name modified (M)OE-PCR, there are five steps: (1) synthesis of individual DNA fragments of interest (with average 20-bp overlap between adjacent fragments) by PCR with high-fidelity pfu DNA polymerase, (2) double-mixing (every two adjacent fragments are mixed to implement OE-PCR without primers), (3) pre-extension (the teams above are mixed to obtain full-length reassembled DNA by OE-PCR without primers), (4) synthesis of the entire DNA of interest by PCR with outermost primers and template DNA from stepxa03, (5) post-extension (ten cycles of PCR at 72°C for annealing and extension are implemented). The method is rapid, simple and error-free. It provides an efficient choice, especially for multiple-site mutagenesis of DNAs; and it can theoretically be applied to the modification of any DNA fragment. Using the MOE-PCR method, we have successfully obtained a modified sam1 gene with eight rare codons optimized simultaneously.

Expression and characterization of a novel highly glucose-tolerant β-glucosidase from a soil metagenome

A β-glucosidase gene unbgl1A was isolated by the function-based screening of a metagenomic library and the enzyme protein was expressed in Escherichia coli, purified, and biochemically characterized. The enzyme Unbgl1A had a Km value of 2.09 ± 0.31 mM, and a Vmax value of 183.90 ± 9.61 μmol min(-1) mg(-1) under the optimal reaction conditions, which were pH 6.0 at 50°C. Unbgl1A can be activated by a variety of monosaccharides, disaccharides, and NaCl, and exhibits a high level of stability at high concentration of NaCl. Two prominent features for this enzyme are: (i) high glucose tolerance. It can be tolerant to glucose as high as 2000 mM, with Ki = 1500 mM; (ii) high NaCl tolerance. Its activity is not affected by 600 mM NaCl. The enzyme showed transglucosylation activities resulting in the formation of cellotriose from cellobiose. These properties of Unbgl1A should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Direct Ethanol Production from Lignocellulosic Sugars and Sugarcane Bagasse by a Recombinant Trichoderma reesei Strain HJ48

Trichoderma reesei can be considered as a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production of T. reesei CICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing.

Characterization of an invertase with pH tolerance and truncation of its N-terminal to shift optimum activity toward neutral pH.

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.

A Novel Acid-Stable Endo-Polygalacturonase from Penicillium oxalicum CZ1028: Purification, Characterization, and Application in the Beverage Industry.

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.

Identification of an acidic endo-polygalacturonase from Penicillium oxalicum CZ1028 and its broad use in major tropical and subtropical fruit juices production

Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum. Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7xa0U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55°C, and was stable between pH 3.0 to 10.0 and below 45°C. The kinetic constants Km and Vmax of PoxaEnPG28A were calculated as 1.57xa0g/L and 14,641.29xa0U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries.

A β-glucosidase from Novosphingobium sp. GX9 with high catalytic efficiency toward isoflavonoid glycoside hydrolysis and (+)-catechin transglycosylation

In view of the important role of isoflavonoids and their related glycoconjugates in human health, there is considerable interest in their enzymatic conversion. SBGL, a novel β-glucosidase isolated from Novosphingobium sp. GX9, was expressed in Escherichia coli and found to have high activity for hydrolysis of daidzin and genistin. SBGL showed very low Km values for daidzin and genistin, and the kcat/Km values for these substrates were 33,300 and 19,200xa0s-1xa0mM-1, respectively. The SBGL glucosidase could also transglycosylate the flavanol (+)-catechin at saturating acceptor concentrations, which has not previously been reported for a β-glucosidase and is difficult to achieve synthetically.

Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans.

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210xa0kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37xa0°C. The apparent Km values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5xa0μM, 10.5xa0mM, 1.3xa0mM, and 5.8xa0mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase.

Identification of a halophilic α-amylase gene from Escherichia coli JM109 and characterization of the recombinant enzyme

A halophilic α-amylase (EAMY) gene from Escherichia coli JM109 was overexpressed in E. coli XL10-Gold and the recombinant protein was purified and characterized. The activity of the EAMY depended on the presence of both Na+ and Cl−, and had maximum activity in 2xa0M NaCl at 55xa0°C and pH 7.0. When 2xa0% (w/v) soluble starch was used as substrate, the specific activity was about 1,090 U mg−1 protein. This is the first report on identifying a halophilic α-amylase with high specific activity from non-halophilic bacteria.

Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process.

An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation inxa0vivo by 56%.