B. amyloliquefaciens

Engineering Substrate Specificity. Although the serine proteases use a common catalytic mechanism, the enzymes have a wide variety of substrate specificities. For example, the natural variant subtiHsins of B. amyloliquefaciens (subtiHsin BPN J and B. licheniformis (subtiHsin Carlsberg) possess very similar stmctures and sequences where 86 of 275 amino acids are identical, but have different catalytic efficiencies, toward tetraamino acid -nitroanilide substrates (67). 

The small 110 a.a. ribonuclease from B. amyloliquefaciens, bamase, has been studied extensively in A.R.Fersht s laboratory (Sali, Bycroft and Fersht, 1988 Matouschek et al, 1990 Bycroft et al., 1990 Serrano and Fersht, 1989) using NMR as an essential experimental techniqne. The df-helices of bamase have been studied with respect to stability and it was found that mutation of the THR and THR residnes located at the N-terminal of the helix (Serrano and Fersht, 1989) could destabilize the protein with up to 2.5 kcal mol b The THR and SER residnes are capable of facilitating the formation of an additional hydrogen bond in the first him of the df-helix. If THR is snbstitnted with ASP or GLU no marked change in stability was observed, probably dne to the known charge-dipole interaction between the negative charge of the ASP or GLU and the positive end of the helix macro-dipole. 

Later, MacGregor and MacGregor16 reported that barley malt a-amylase had an action pattern that was similar to that of B. amyloliquefaciens a-amylase, forming... 

Using molecular biology techniques, Conrad et al.61 produced hybrids of the a-amylases from B. amyloliquefaciens and B. licheniformis. Thirty-three hybrids were formed. They consisted of the entire a-amylase sequence with variable proportions from B. amyloliquefaciens a-amylase and B. licheniformis a-amylase. The hybrid enzymes fell into six groups that retained the extra-thermostability of the licheniformis enzyme. A specific hybrid sequence (residues 34-76) was correlated with the enzymes product specificity for forming and accumulating maltohexaose. Two of the hybrids were less thermostable than either of the parent types, while two others were enzymatically inactive. 

Kj values, however, were in the nanomolar range, with the Kj for the maltohexaosyl acarbose, 33 nM for A. oryzae a-amylase, 37 nM for B. amyloliquefaciens a-amylase, 14nM for human salivary a-amylase and 7.0nM for porcine pancreatic a-amylase. These inhibition constants represented relative inhibitory potency over acarbose of 8200-, 351 -, 90- and 114-times, respectively for the four a-amylases.220d... 

In December 1988, the company introduced a new strain of B. amyloliquefaciens (strain V), which had been genetically modified to increase the synthesis of 5-phosphoribosyl-l-pyrophosphate, an intermediate in the biosynthesis of tryptophan (see Figure 1). After fermentation, tryptophan was extracted from the broth and purified using a series of filtration, crystallization, and separation processes. The purification procedures included contact with powdered activated carbon and then granulated activated carbon. The amount of powdered activated carbon in each batch was usually... 

B. amyloliquefaciens Asp32 D32A Reduction of kcat by factors Importance of catalytic triad, D32,... 

Many of today s industrial enzymes are produced hy Bacillus species, especially B. subtilis, B. amyloliquefaciens, and B. licheniformis. These include amylases. 

Curie-point pyrolysis MS was applied to the identification of selected Bacillus species by Shute et al. Fifty-three strains of B. subtilis, B. pumilus, B. licheniformis, and B. amyloliquefaciens in both a spomlating and nonspomlating state were pyro-... 

Stephens, M.A., Ortlepp, S.A., Ollington, J.F. and McConnell, D.J., 1984, Nucleotide sequence of the 5 region of the Bacillus llcheniformis a-amylase gene comparison with the B. amyloliquefaciens gene, J. Bacterlol., 158 369. 

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