Computer-Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns

A main remaining challenge in protein engineering is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. This limits researchers in exploiting nature's potential in generating better enzymes. The Computer-assisted Recombination (CompassR) strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔG_fold). The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of Bacillus subtilis lipase A. The finally obtained variant F17S/V54K/D64N/D91E had a 2.7-fold improved specific activity in 18.3 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). In essence, the deducted CompassR rule allows recombination of beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner.     

碱性蛋白酶高产菌株的选育

以利福平抗性及牛奶平板上产生的透明水解圈的大小作为选择标记，用钴60对本实验室筛选获得的碱性蛋白酶高产菌株地衣芽孢杆菌C6进行诱变处理，筛选获得了一株酶产量大幅度提高的菌株L106，经发酵条件优化，其产酶活性稳定在19000U／mL左右，是出发菌株的1．9倍．与出发菌株相比，该菌的发酵周期也有所缩短，而产酶的最适温度和最适pH保持不变，并保持了芽孢形成较少的优良特性，具有良好的实际应用潜力。     

Surfactin-like structures of five cyclic depsipeptides from the marine isolate ofBacillus pumilus

Five cyclic depsipeptides with molecular masses of 1007, 1021, 1021, 1035, and 1035 were obtained fromBacillus pumilus KMM 150 associated with Australian marine spongeIrcinia sp. Their structures were assigned by mass spectrometric techniques (high-resolution fast atom bombardment and electron impact mass spectrometry), chemical modification, and extensive spectroscopic analysis, including several types of two-dimensional NMR.Deceased.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 979–983, May, 1995.The authors are grateful to Dr. V. V. Mikhailov, the Head of the Laboratory of marine microbiology of PIBC of the Far-Eastern Branch of the RAS, and to Dr. E. P. Ivanova for isolation, systematic determination, and cultivation of the strain KMM 150. The authors are also thankful to T. I. Zykova for carrying out the fermentative hydrolysis of peptide4c.     

Antiglioma pseurotin A from marine Bacillus sp. FS8D regulating tumour metabolic enzymes

Pseurotin A was isolated from a culture of marine Bacillus sp. FS8D and showed to be active against the proliferation of four different glioma cells with IC₅₀ values of 0.51–29.3 μM. It has been found that pseurotin A downregulated the expression of tumour glycolytic enzymes pyruvate kinase M2 (PKM2) and lactate dehydrogenase 5 (LDH5) and upregulated the expression of pyruvate dehydrogenase beta (PDHB), adenosine triphosphate synthase beta (ATPB) and cytochrome C (Cyto-C), the important regulators for tricarboxylic acid cycle and oxidative phosphorylation. The data suggested that targeting multiple metabolic enzymes might be one of the antiglioma mechanisms of pseurotin A.     

Purification and characterization of theD-hydantoinase from bacillus circulans

AD-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity fromBacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0–10.0 and a temperature of 75‡C. The enzyme is the most stable in a pH range of 8.5–9.5 and up to 60‡C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Natural and edible biopolymer poly-gamma-glutamic acid: synthesis, production, and applications

Poly-gamma-glutamic acid (gamma-PGA) is a very promising biodegradable polymer that is produced by Bacillus subtilis. Gamma-PGA is water-soluble, anionic, biodegradable, and edible. This paper reviews the production of a strain of gamma-PGA and recent developments with respect to applications in terms of Ca absorption, moisturizing properties, gamma-PGA conjugation, super absorbent polymer, and so on. Our recent research shows that gamma-PGA can be used as an immune-stimulating and anti-tumor agent, especially at high molecular weight.     

A novel synthesis method for cyclodextrins from maltose in water-organic solvent systems

A novel enzymatic synthesis method of cyclodextrin (CD) from low-mol-wt maltose using cyclomaltodextrin glucanotransferase (CGTase) fromBacillus macerans has been developed in various water-organic solvent systems. A Β-CD was synthesized in a two-phase system consisting of water and cyclohexane. However, no CDs could be synthesized in an aqueous buffer solution. A maximal yield of Β- CD has been obtained at a cyclohexane content volume of 44%. This synthesis has been obtained only at low temperatures, i.e., 7‡C, and did not take place at 50‡C. In addition, various organic solvents have been used for the enzymatic synthesis of CD from maltose. Consequently, Β-CD could be synthesized in various water-organic solvent systems, e.g., cyclohexane, benzene, xylene, and chloroform, but no enzymatic reaction occurred using aliphaticn-hydrocarbon solvents such as hexane, dodecane, and hexadecane. Furthermore, α- and Β- CD could be synthesized in water mixture solutions using organic solvents having an alcoholic group (e.g., ethanol, propanol, butanol, and pentanol) in a wide range of the reaction temperatures, typically 7–50‡C. In this temperature range, α- and Β-CD were also formed and the maximal yield from maltose to Β-CD of approx 13% was reached in 60 h.     

Specificity and mode of action of a thermostable xylanase from bacillus amyloliquefaciens on-line monitoring of hydrolysis products

A thermostable xylanase purified from a strain of Bacillus amyloliquefaciens MIR 32 was characterized with respect to its substrate specificity and mode of hydrolytic action. The enzyme was highly specific for xylans as substrate and displayed no activity toward other polysaccharides, including cellulose. The enzyme exhibited K_m and V_max of 4.5 mg/mL and 0.58 mmol/min/mg, respectively, with birchwood xylan as the substrate. Microdialysis sampling with anion exchange chromatography and integrated pulsed electrochemical detection were used for rapid on-line monitoring of products during hydrolysis of oat spelt and bagasse xylan, and xylooligosaccharides. Xylobiose and xylotriose were the main end products. Xylotetraose was the smallest oligosaccharide to be acted on by the xylanase. The product pattern confirmed that the enzyme was an endoxylanase.     

growth kinetics ofbacillus stearothermophilus BR219

Bacillus stearothermophilus BR219, a phenol-resistant thermophile, can convert phenol to the specialty chemical catechol. The growth kinetics of this organism were studied in batch, continuous, and im-mobilized-cell culture. Batch growth was insensitive to pH between 6.0 and 8.0, but little growth occurred at 5.5. In continuous culture on a dilute medium supplemented with 10 mM phenol, several steady states were achieved between dilution rates of 0.25 and 1.3 h^-1. Phenol degradation was found to be uncoupled from growth. Immobilized cells grew rapidly in a rich medium, but cell viability plummeted following a switch to a dilute medium supplemented with 5 mM phenol.     

The metabolism of aromatic compounds by thermophilicBacilli

A range of thermophilicBacilli were screened for the ability to grow on aromatic compounds. Five out of ten of those studied were able to utilize aromatic acids as a sole carbon source. OtherBacilli were purified after enrichment on aromatic compounds. One of these isolates, a strain ofBacillus stearothermophilus, degraded both phenol and benzoic acid. Phenol degradation proceeded via catechol, and thereafter by oxidative and nonoxidativemeta-cleavage routes. The catalytic properties of cell-free extracts displaying the activities of the initial oxygenases have been described.